trichostatin-a and Breast-Neoplasms

Topics: Animals; Antineoplastic Agents; Biogenic Polyamines; Breast Neoplasms; DNA; Drug Discovery; Eflornithine; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Demethylases; Homeostasis; Humans; Oxidoreductases, N-Demethylating; Polyamines; Structure-Activity Relationship

The aim of this study was to sensitize the resistant breast adenocarcinoma cells towards Tumour Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced apoptosis.. Breast cancer is a heterogeneous disease involving complex mechanisms. TRAIL is a potential anticancer candidate for targeted treatment due to its selective killing effects on neoplastic cells. Nonetheless, resistance occurs in many cancers either intrinsically or after multiple treatments.. Therefore, this research investigated whether the combination of Trichostatin A (TSA) and Zebularine (Zeb) (TZ) followed by TRAIL (TZT) could sensitize the human breast adenocarcinoma cells towards apoptosis.. The breast adenocarcinoma cells, MDA-MB-231, MCF-7 and E-MDA-MB-231 (E-cadherin re-expressed MDA-MB-231) were treated with TSA, Zeb, TZ, TRAIL and TZT. The cells were subjected to hematoxylin and eosin (H & E) staining and FITC-Annexin V/Propidium Iodide apoptosis detection prior to proteome profiling.. Based on morphological observation, apoptosis was induced in all cells treated with all treatment regimens though it was more evident for the TZT-treated cells. In the apoptosis detection analysis, TZ increased early apoptosis significantly in MDA-MB-231 and MCF-7 while TRAIL induced late apoptosis significantly in E-MDA-MB-231. Based on the proteome profiling on MDA-MB-231, TRAIL R2 and Fas expression was increased. For E-MDA-MB- 231, down-regulation of catalase, paraoxonase-2 (PON2), clusterin, an inhibitor of apoptosis proteins (IAPs) and cell stress proteins validated the notion that E-cadherin re-expression enhances TZT anti-cancer efficacy. Similar trend was observed in MCF-7 whereby TZT treatment down-regulated the anti-apoptotic catalase and PON2, increased the proapoptotic, B cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad) and Bcl-2-associated X (Bax), second mitochondria-derived activator of caspase (SMAC) and HtrA serine peptidase 2 (HTRA2) as well as TRAIL receptors (TRAIL R1 and TRAIL R2).. TZ treatment serves as an efficient treatment regimen for MDA-MB-231 and MCF-7, while TRAIL serves as a better treatment option for E-MDA-MB-231. Therefore, future studies on E-cadherin's positive regulatory role in TRAIL-induced apoptosis are warranted.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Cadherins; Catalase; Cell Line, Tumor; Female; Humans; Inhibitor of Apoptosis Proteins; Ligands; Proteome; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Compact chromatin is linked to a poor tumour prognosis and resistance to radiotherapy from photons. We investigated DNA damage induction and repair in the context of chromatin structure for densely ionising alpha radiation as well as its therapeutic potential. Chromatin opening by histone deacetylase inhibitor trichostatin A (TSA) pretreatment reduced clonogenic survival and increased γH2AX foci in MDA-MB-231 cells, indicative of increased damage induction by free radicals using gamma radiation. In contrast, TSA pretreatment tended to improve survival after alpha radiation while γH2AX foci were similar or lower; therefore, an increased DNA repair is suggested due to increased access of repair proteins. MDA-MB-231 cells exposed to fractionated gamma radiation (2 Gy × 6) expressed high levels of stem cell markers, elevated heterochromatin H3K9me3 marker, and a trend towards reduced clonogenic survival in response to alpha radiation. There was a higher level of H3K9me3 at baseline, and the ratio of DNA damage induced by alpha vs. gamma radiation was higher in the aggressive MDA-MB-231 cells compared to hormone receptor-positive MCF7 cells. We demonstrate that heterochromatin structure and stemness properties are induced by fractionated radiation exposure. Gamma radiation-exposed cells may be targeted using alpha radiation, and we provide a mechanistic basis for the involvement of chromatin in these effects.

Topics: Acetylation; Alpha Particles; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Clone Cells; Female; Gamma Rays; Heterochromatin; Histones; Humans; Hydroxamic Acids; Lysine; Neoplastic Stem Cells; Radiation Exposure; Spheroids, Cellular

To elucidate the role of histone deacetylase inhibitor Trichostatin A (TSA) in affecting metastasis of breast carcinoma, and its molecular mechanism.. LPAR5 levels in breast carcinoma tissues and paracancerous tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR), and its expression pattern was further verified in breast carcinoma cell lines. The relationship between LPAR5 and prognosis of breast carcinoma patients was analyzed. After TSA induction (100-400 nmol/L) for 6-48 h, the proliferative and migratory abilities of SKBR3 and MDA-MB-231 cells in overexpressing LPAR5 were examined by cell counting kit-8 (CCK-8), transwell and wound healing assay. By constructing a xenograft model in nude mice, the influences of TSA and LPAR5 on in vivo growth of breast carcinoma were examined.. LPAR5 was upregulated in breast carcinoma samples. High level of LPAR5 predicted higher rates of lymphatic metastasis and distant metastasis, as well as lower overall survival and progression-free survival in breast carcinoma patients. LPAR5 level was dose-dependently downregulated in TSA-induced SKBR3 and MDA-MB-231 cells. In addition, TSA induction dose-dependently declined proliferative ability, and time-dependently attenuated migratory ability in breast carcinoma cells. In vivo overexpression of LPAR5 in nude mice reversed the inhibitory effect of TSA on breast carcinoma growth.. TSA induction can suppress proliferative and migratory abilities in breast carcinoma by downregulating LPAR5.

Topics: Animals; Breast Neoplasms; Cell Line; Cell Movement; Dose-Response Relationship, Drug; Down-Regulation; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Middle Aged; Receptors, Lysophosphatidic Acid

Some drugs can be used to treat multiple diseases, suggesting potential patterns in drug treatment. Determination of drug treatment patterns can improve our understanding of the mechanisms of drug action, enabling drug repurposing. A drug can be associated with a multilayer tissue-specific protein-protein interaction (TSPPI) network for the diseases it is used to treat. Proteins usually interact with other proteins to achieve functions that cause diseases. Hence, studying drug treatment patterns is similar to studying common module structures in multilayer TSPPI networks. Therefore, we propose a network-based model to study the treatment patterns of drugs. The method was designated SDTP (studying drug treatment pattern) and was based on drug effects and a multilayer network model. To demonstrate the application of the SDTP method, we focused on analysis of trichostatin A (TSA) in leukemia, breast cancer, and prostate cancer. We constructed a TSPPI multilayer network and obtained candidate drug-target modules from the network. Gene ontology analysis provided insights into the significance of the drug-target modules and co-expression networks. Finally, two modules were obtained as potential treatment patterns for TSA. Through analysis of the significance, composition, and functions of the selected drug-target modules, we validated the feasibility and rationality of our proposed SDTP method for identifying drug treatment patterns. In summary, our novel approach used a multilayer network model to overcome the shortcomings of single-layer networks and combined the network with information on drug activity. Based on the discovered drug treatment patterns, we can predict the potential diseases that the drug can treat. That is, if a disease-related protein module has a similar structure, then the drug is likely to be a potential drug for the treatment of the disease.

Topics: Breast Neoplasms; Drug Repositioning; Female; Gene Ontology; Gene Regulatory Networks; Humans; Hydroxamic Acids; Leukemia; Male; Models, Biological; Prostatic Neoplasms; Protein Interaction Maps; Protein Synthesis Inhibitors; Transcriptome

Histone deacetylase inhibitors (HDACis) are able to suppress breast cancer cells in vitro and in vivo by altering the expression of estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor 2 (Her2/neu). Since HDACis can alter the expression of various microRNAs (miRNAs/miRs), the present study aimed to examine the role of miRNAs in the effects of HDACis on breast cancer cells. We first examined the mRNA expression of ER, PR, and Her2/neu using RT-PCR and the protein levels of ER, PR, and Her2/neu using western blot analysis in MDA-MB-231 and BT474 cells, after trichostatin A (TSA) or vorinostat (SAHA) treatment. We then conducted miRNA expression profiling using microarrays after BT474 cells were treated with TSA or SAHA. Finally, we examined the effects of synthetic miR-762 and miR-642a-3p inhibitors on SAHA-induced downregulation of Her2/neu and SAHA-induced apoptosis and PARP cleavage in BT474 cells. The results indicated that TSA and SAHA dose‑dependently enhanced the mRNA and protein expression levels of ER and PR in MDA‑MB‑231 and BT474 cells. In addition, the mRNA expression levels of Her2/neu were reduced in MDA‑MB‑231 cells, and the mRNA and protein expression levels of Her2/neu were reduced in BT474 cells in response to SAHA and TSA. Furthermore, treatment with TSA (0.2 µM) or SAHA (5.0 µM) induced a marked alteration in the expression of various miRNAs in BT474 cells. Notably, when cells were cotransfected with miR‑762 and miR‑642a‑3p inhibitors, SAHA‑induced downregulation of Her2/neu was inhibited, and SAHA‑induced apoptosis and poly (ADP‑ribose) polymerase cleavage were significantly reduced in BT474 cells. These results indicated that numerous HDACi‑induced miRNAs are required to downregulate Her2/neu levels and promote apoptosis of Her2‑overexpressing breast cancer cells.

Topics: Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MicroRNAs; RNA, Messenger; Transfection; Vorinostat

Acquired resistance against doxorubicin is a major limitation in clinical treatment of breast cancer. The molecular mechanism behind the aberrant expression of genes leading to doxorubicin resistance is not clear. Epigenetic changes play an important role in the regulation of gene expression. Therefore, the objective of this study was to identify the epigenetic mechanism underlying acquired doxorubicin resistance in breast cancer cells. Doxorubicin-resistant cells were selected by repeated exposure of MCF-7 and MDA-MB-231 breast cancer cell lines to clinically relevant doses of doxorubicin for 18 months. MTT assay, cell cycle analysis, colony formation, qRT-PCR, and Western blot analyses were used to characterize the epigenetic and molecular mechanism. Pyrosequencing was used to detect MSH2 promoter hypermethylation. Aberrant expression of epigenetic regulatory genes, a significant increase in H3 acetylation and methylation, as well as promoter hypermethylation-mediated inactivation of MSH2 gene were associated with the acquired resistant phenotype. Demethylating agent 5-Aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly re-sensitized resistant cells to doxorubicin. Findings of this study revealed that epigenetic aberrations including promoter hypermethylation-mediated inactivation MSH2 contribute to the acquisition of doxorubicin resistance in breast cancer cells. Additionally, our data suggest that some of these epigenetic aberrations are progressive during resistance development and therefore can potentially be used as biomarkers for early detection of resistance. These epigenetic aberrations, being reversible, can also serve as targets for epigenetic therapy to re-sensitize doxorubicin-resistant breast cancer cells. Epigenetic inactivation of mismatch repair gene MSH2 further suggests that loss of MMR-dependent apoptotic potential could be a novel mechanistic basis for the acquisition of doxorubicin resistance in breast cancer cells.

Topics: Breast Neoplasms; Doxorubicin; Drug Resistance, Neoplasm; Epigenesis, Genetic; Epigenomics; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MCF-7 Cells

Identifying markers for breast cancer is important for both diagnosis and the design of treatment strategies. Recent studies have implicated Paired box 5 (

Topics: Animals; Apoptosis; Aza Compounds; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA Methylation; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; MCF-7 Cells; Mice; PAX5 Transcription Factor; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Wound Healing

Chemotherapy is the most common clinical option for treatment of breast cancer. However, the efficacy of chemotherapy depends on the age of breast cancer patients. Breast tissues are estrogen responsive and the levels of ovarian estrogen vary among the breast cancer patients primarily between pre- and post-menopausal age. Whether this age-dependent variation in estrogen levels influences the chemotherapeutic efficacy in breast cancer patients is not known. Therefore, the objective of this study was to evaluate the effects of natural estrogen 17 beta-estradiol (E2) on the efficacy of chemotherapeutic drugs in breast cancer cells. Estrogen responsive MCF-7 and T47D breast cancer cells were long-term exposed to 100 pg/ml estrogen, and using these cells the efficacy of chemotherapeutic drugs doxorubicin and cisplatin were determined. The result of cell viability and cell cycle analysis revealed increased sensitivities of doxorubicin and cisplatin in estrogen-exposed MCF-7 and T47D cells as compared to their respective control cells. Gene expression analysis of cell cycle, anti-apoptosis, DNA repair, and drug transporter genes further confirmed the increased efficacy of chemotherapeutic drugs in estrogen-exposed cells at molecular level. To further understand the role of epigenetic mechanism in enhanced chemotherapeutic efficacy by estrogen, cells were pre-treated with epigenetic drugs, 5-aza-2-deoxycytidine and Trichostatin A prior to doxorubicin and cisplatin treatments. The 5-aza-2 deoxycytidine pre-treatment significantly decreased the estrogen-induced efficacy of doxorubicin and cisplatin, suggesting the role of estrogen-induced hypermethylation in enhanced sensitivity of these drugs in estrogen-exposed cells. In summary, the results of this study revealed that sensitivity to chemotherapy depends on the levels of estrogen in breast cancer cells. Findings of this study will have clinical implications in selecting the chemotherapy strategies for treatment of breast cancer patients depending on the serum estrogen levels that varies among pre- and post-menopausal age of the patients.

Topics: Antineoplastic Agents; Apoptosis; Azacitidine; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cisplatin; Decitabine; DNA Methylation; Doxorubicin; Drug Synergism; Epigenesis, Genetic; Estradiol; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; MCF-7 Cells

Breast cancer is the most common cancer type and cause of cancer-related mortality among women worldwide. New biomarker discovery is crucial for diagnostic innovation and personalized medicine in breast cancer. Heat shock proteins (HSPs) have been increasingly reported as biomarkers and potential drug targets for cancers. HLJ1 (DNAJB4) belongs to the DNAJ (HSP40) family of HSPs and is regarded as a tumor suppressor gene in lung, colon, and gastric cancers. However, the role of the HLJ1 gene in breast cancer is currently unknown. We evaluated the role of the HLJ1 gene in breast cancer progression by analyzing its in vitro and in vivo expression and its genetic/epigenetic alterations. HLJ1 expression was found to be reduced or lost in breast cancer cell lines (SK-BR-3, MDA-MB-231, ZR-75-1) compared with the nontumorigenic mammary epithelial cell line (MCF 10A). In a clinical context for breast cancer progression, the HLJ1 expression was significantly less frequent in invasive breast carcinoma samples (n = 230) compared with normal breast tissue (n = 100), benign neoplasia (n = 53), and ductal carcinoma in situ (n = 21). In methylation analyses by the combined bisulfite restriction analysis assay, the CpG island located in the 5'-flanking region of the HLJ1 gene was found to be methylated in breast cancer cell lines. HLJ1 expression was restored in the ZR-75-1 cell line by DNA demethylating agent 5-Aza-2'-deoxycytidine (5-AzadC) and histone deacetylase inhibitor trichostatin A. These new observations support the idea that HLJ1 is a tumor suppressor candidate and potential biomarker for breast cancer. Epigenomic mechanisms such as CpG methylation and histone deacetylation might contribute to downregulation of HLJ1 expression. We call for future functional, epigenomic, and clinical studies to ascertain the contribution of HLJ1 to breast cancer pathogenesis and, importantly, evaluate its potential for biomarker development in support of personalized medicine diagnostic innovation in clinical oncology.

Topics: Adult; Azacitidine; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cohort Studies; CpG Islands; Decitabine; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; HSP40 Heat-Shock Proteins; Humans; Hydroxamic Acids; Kaplan-Meier Estimate; Precision Medicine; RNA, Messenger

Inhibition of histone deacetylase enzymes (HDACs) has been well documented as an attractive target for the development of chemotherapeutic drugs. The present study investigated the effects of two prototype hydroxamic acid HDAC inhibitors, namely Trichostatin A (TSA) and Belinostat (PXD‑101) and the benzamide Entinostat (MS‑275) in A2780 ovarian carcinoma and MCF7 breast adenocarcinoma cells. The three HDACi inhibited the proliferation of A2780 and MCF7 cells at comparable levels, below the µM range. Enzyme inhibition assays in a cell‑free system showed that TSA was the most potent inhibitor of total HDAC enzyme activity followed by PXD‑101 and MS‑275. Incubation of A2780 and MCF7 cells with the hydroxamates TSA and PXD‑101 for 24 h resulted in a dramatic increase of acetylated tubulin induction (up to 30‑fold for TSA). In contrast to acetylated tubulin, western blot analysis and flow cytometry indicated that the induction of acetylated histone H4 was considerably smaller. The benzamide MS‑275 exhibited nearly a 2‑fold induction of acetylated histone H4 and an even smaller induction of acetylated tubulin in A2780 and MCF7 cells. Taken together, these data suggest that although the three HDACi were equipotent in inhibiting proliferation of MCF7 and A2780 cells, only the benzamide MS‑275 did not induce acetylated tubulin expression, a marker of class IIb HDACs.

Topics: Acetylation; Benzamides; Breast Neoplasms; Cell Proliferation; Cell-Free System; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; MCF-7 Cells; Ovarian Neoplasms; Protein Processing, Post-Translational; Sulfonamides; Tubulin

Molecule-targeted therapy has achieved great progress in cancer therapy. Effective drug combinations are one way to enhance the therapeutic efficacy and combat resistance. Here, we determined the effect of the PI3K/mTOR dual inhibitor BEZ235 and the histone deacetylase inhibitor Trichostatin A (TSA) on human breast cancer. We demonstrated that the combination of BEZ235 and TSA results in significant synergistic growth inhibition of multiple breast cancer cell lines. Mechanistic studies revealed that the combined therapy induced apoptosis in a caspase-dependent manner, which might be related to the further depression of the PI3K/Akt/mTOR signalling pathway. Additionally, co-treatment with BEZ235 and TSA enhanced autophagic cell death by up-regulating the expression of LC3B-II and Beclin-1. The vivo tumour modelling studies revealed that BEZ235 combined with TSA blocked tumour growth without noticeable side effects. These data suggest that the combination of BEZ235 and TSA may be a new selective strategy, which may have significant clinical application in the treatment of breast cancer patients.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Imidazoles; MCF-7 Cells; Mice; Mice, Nude; Phosphoinositide-3 Kinase Inhibitors; Quinolines; Signal Transduction; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

In breast carcinoma cells, high levels of hyaluronan (HA) and its CD44 receptor are frequently associated with alteration in estrogen signaling. We demonstrate that stable hyaluronate synthase 2 (HAS2) overexpression in estrogen receptor α (ERα) -positive MCF7 cells oppositely altered estrogen dependence of cell growth and its sensitivity towards antiestrogens. Albeit without effect on ERα expression and estradiol binding properties, HAS2 overexpression increased ERα Ser118 phosphorylation as well as transcriptional activity of estrogen in an ERE-luciferase reporter gene assay. However, HAS2 overexpression induced partial silencing of E2 driven-genes without affecting the magnitude of regulation by estradiol. This effect was associated with half-reduction in the activity of nuclear histone deacetylases (HDACs) through a post-translational mechanism likely consecutive to the enhanced expression of the histone acetyl-transferase EP300. In conclusion, increase in HA/CD44 interactions may contribute, through an HDAC inhibitor-like and ER-independent mechanism, to the silencing of estrogen-driven genes in breast carcinoma.

Topics: Acetylation; Breast Neoplasms; E1A-Associated p300 Protein; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glucuronosyltransferase; Histone Deacetylase Inhibitors; Humans; Hyaluronan Receptors; Hyaluronan Synthases; Hyaluronic Acid; Hydroxamic Acids; MCF-7 Cells; Methylation; Phosphorylation; Phosphoserine; Protein Binding; Subcellular Fractions

Recent studies have shown that expression of Sarco(endo)plasmic Reticulum Ca(2+) -ATPase 2 (SERCA2) is decreased in oral cancer; whereas expression of SERCA3 is considerably decreased or absent in human colon, gastric, breast, and lung cancers. The ATP2A2 and ATP2A3 genes encode SERCA2 and SERCA3 isoforms, respectively. Promoter methylation on CpG islands was responsible for the repression of ATP2A2 gene in human oral cancer samples. On the other hand, histone deacetylase inhibitors (HDACi) up-regulate ATP2A3 expression in gastric, colon, and lung cancer cells in culture, however, the molecular mechanism is unknown. In this study, we investigate whether HDACi and DNA methylation regulate ATP2A2 and ATP2A3 expression in human breast cancer cell lines. Results show a marked induction of SERCA3a and pan-SERCA3 mRNA expression in human MCF-7 and MDA-MB-231 cells treated with sodium butyrate (NaB) or trichostatin A (TSA); whereas SERCA2b mRNA expression did not change significantly. ChIP assays show that NaB or TSA treatment of MDA-MB-231 cells increases H3K9 acetylation on ATP2A3 promoter. NaB also decreases H3K9 trimethylation; suggesting that these modifications stimulate ATP2A3 gene expression, through a chromatin remodeling mechanism. In contrast, NaB or TSA do not increase H3K9-acetylation of ATP2A2 proximal promoter. In addition, treatment with 5-aza-2'-deoxycytidine did not affect SERCA2b and SERCA3a expression, suggesting that promoter methylation status does not alter their expression in these cell lines. We propose that alteration of SERCA2b/SERCA3a isoform expression ratio could affect calcium management within the cell, and thus, the cellular pathways regulated by calcium could be compromised, such as cellular proliferation or apoptosis. © 2015 Wiley Periodicals, Inc.

Topics: Breast Neoplasms; Butyric Acid; Cell Line, Tumor; CpG Islands; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MCF-7 Cells; Promoter Regions, Genetic; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Up-Regulation

Phenylbutyrate (PB) is a histone deacetylase antagonist that also exhibits antitumor activity. In this study, we used 7 breast cancer cell lines to identify biomarker candidates that predict PB sensitivity in breast cancer.Comprehensive gene expression profiles were compared using microarrays, and the importance of the identified genes to PB sensitivity was confirmed in gene transfection experiments. CRL and MDAMB453 cells were identified as PB-sensitive, while MDAMB231 cells were PB-resistant.RAB25 and ESRP1 were identified as key regulators of PB sensitivity, while ANKD1, ETS1, PTRF, IFI16 and KIAA1199 acted as PB resistance-related genes. Expression of these genes was dramatically altered by DNA demethylation treatments. RAB25 expression inhibited IFI16 and PTRF, while ESRP1 expression suppressed ANKRD1, ETS1, and KIAA1199. Both RAB25 and ESRP1 were suppressed by ZEB1, which was in turn regulated via epigenetic mechanisms. Thus, PB sensitivity is influenced by epigenetic expression alteration of ZEB1. The genes associated with PB sensitivity are downstream targets of ZEB1. Epigenetic regulation of ZEB1 may prove valuable as a critical biomarker for predicting resistance to breast cancer therapies.

Topics: Antineoplastic Agents; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Decitabine; Drug Resistance, Neoplasm; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Immunohistochemistry; MCF-7 Cells; Phenylbutyrates; rab GTP-Binding Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; Signal Transduction; Trastuzumab; Zinc Finger E-box-Binding Homeobox 1

Estrogen receptor (ER) is a crucial determinant of resistance to endocrine therapy, which may change during the progression of breast cancer. We previously showed that hypoxia induces ESR1 gene repression and ERα protein degradation via proteasome-mediated pathway in breast cancer cells. HDAC plays important roles in the regulation of histone and non-histone protein post-translational modification. HDAC inhibitors can induce epigenetic changes and have therapeutic potential for targeting various cancers. Trichostatin A exerts potent antitumor activities against breast cancer cells in vitro and in vivo. In this report, we show that TSA augments ESR1 gene repression at the transcriptional level and downregulates ERα protein expression under hypoxic conditions through a proteasome-mediated pathway. TSA-induced estrogen response element-driven reporter activity in the absence of estrogen was synergistically enhanced under hypoxia; however, TSA inhibited cell proliferation under both normoxia and hypoxia. Our data show that the hypoxia-induced repression of ESR1 and degradation of ERα are enhanced by concomitant treatment with TSA. These findings expand our understanding of hormone responsiveness in the tumor microenvironment; however, additional in-depth studies are required to elucidate the detailed mechanisms of TSA-induced ERα regulation under hypoxia.

Topics: Breast Neoplasms; Cell Hypoxia; Down-Regulation; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; MCF-7 Cells; Oxygen

Claudin-6 (CLDN6), a member of claudin transmembrane protein family, has recently been reported to be undetectable or at low levels in human breast cancer cell lines and tissues and plays a role in suppression of migration and invasion in breast cancer cells. In addition, it is reported that CLDN6 expression is regulated by DNA methylation in various human cancers and cell lines. However, it is unclear how DNA methylation regulates CLDN6 expression. Here we show the mechanism by which DNA methylation regulates CLDN6 expression in human breast cancer cell line MCF-7.. RT-PCR, Western blot and immunofluorescent staining were utilized to investigate CLDN6 expression in breast cancer tissues and MCF-7 cells. Methylation-Specific PCR (MSP) was applied to determine DNA methylation status in CLDN6 gene promoter region. Wound-healing assay and invasion assay were utilized to test mobility of MCF-7 cells treated with 5-aza-dC (DNA methyltransferase inhibitor). MeCP2 binding, H3Ac and H4Ac in CLDN6 promoter region were analyzed by ChIP assay. Nuclease accessibility assay was performed for analysis of the chromatin conformation of CLDN6 gene. To study the role of CLDN6 in malignant progression, we used RNAi to knockdown CLDN6 expression in MCF-7 cells treated with 5-aza-dC, and examined the mobility of MCF-7 cells by wound-healing assay and invasion assay.. 5-aza-dC and TSA (histone deacetylase inhibitor) application induced CLDN6 expression in MCF-7 cells respectively and synergistically. 5-aza-dC treatment induced CLDN6 demethylation, inhibited MeCP2 binding to CLDN6 promoter and increased H3Ac and H4Ac in the promoter. In addition, TSA increased H4Ac, not H3Ac in the promoter. The chromatin structure of CLDN6 gene became looser than the control group after treating with 5-aza-dC in MCF-7 cells. 5-aza-dC up-regulated CLDN6 expression and suppressed migration and invasion in MCF-7 cells, whereas CLDN6 silence restored tumor malignance in MCF-7 cells.. DNA methylation down-regulates CLDN6 expression through MeCP2 binding to the CLDN6 promoter, deacetylating H3 and H4, and altering chromatin structure, consequently promoting migratory and invasive phenotype in MCF-7 cells.

Topics: Acetylation; Adult; Aged; Aged, 80 and over; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Claudins; DNA Methylation; Epigenesis, Genetic; Female; Histones; Humans; Hydroxamic Acids; MCF-7 Cells; Methyl-CpG-Binding Protein 2; Middle Aged; Neoplasm Invasiveness; Promoter Regions, Genetic; Young Adult

Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs), using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA) and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP), caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells.

Topics: Apoptosis; Breast Neoplasms; Caspase 3; Cell Survival; Enzyme Activation; Female; HeLa Cells; Histone Deacetylases; Humans; Hydroxamic Acids; Membrane Potential, Mitochondrial; Nanoparticles; Oxidative Stress; Palladium; Uterine Cervical Neoplasms

Tumor cells display different bioenergetic profiles when compared to normal cells. In the present work we showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin A in breast cancer cells representing different stages of aggressiveness and metabolic profile. When testing the effect of NaB and TSA on viability of cells, it was shown that non-tumorigenic MCF-10A cells were less affected by increasing doses of the drugs than the tumorigenic, hormone dependent, tightly cohesive MCF-7, T-47D and the highly metastatic triple-negative MDA-MB 231 cells. T-47D cells were the most sensitive to treatment with both, NaB and TSA. Experiments measuring anchorage- independent growth of tumor cells showed that MCF-7, T-47D, and MDA-MB-231 cells were equally sensitive to the treatment with NaB. The NaB induced an attenuation of glycolysis, reflected by a decrease in lactate release in MCF-7 and T47D lines. Pyruvate kinase activity was significantly enhanced by NaB in MDA-MB-231 cells only. In contrast, the inhibitor enhanced lactate dehydrogenase activity specifically in T-47 D cells. Glucose-6-phosphate dehydrogenase activity was shown to be differentially modulated by NaB in the cell lines investigated: the enzyme was inhibited in MCF-7 cells, whereas in T-47D and MDA-MB-231 cells, G6PDH was activated. NaB and TSA were able to significantly increase the oxygen consumption by MDA-MB-231 and T-47D cells. Collectively the results show that epigenetic changes associated to acetylation of proteins in general affect the energy metabolism in all cancer cell lines and that mitochondria may occupy a central role in metastasis.

Topics: Breast Neoplasms; Butyric Acid; Cell Line, Tumor; Energy Metabolism; Glycolysis; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Metabolic Networks and Pathways; Oxidation-Reduction

Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.

Topics: 5' Flanking Region; Acetylation; Base Sequence; Binding Sites; Breast Neoplasms; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation; Electrophoretic Mobility Shift Assay; Female; Histones; Humans; Hydroxamic Acids; Inorganic Pyrophosphatase; MCF-7 Cells; Molecular Sequence Data; Mutagenesis, Site-Directed; p300-CBP Transcription Factors; Promoter Regions, Genetic; RNA Interference; RNA, Small Interfering; Sp1 Transcription Factor; Transcriptional Activation; Up-Regulation

The present study evaluated the mechanism of apoptosis caused by post-translational modification, hyperacetylation in triple-negative breast cancer (TNBC) cells. We previously showed that trichostatin A (TSA) induced secretion of acetylated apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1). This is the first report showing that Ac-APE1/Ref-1 initiates apoptosis in TNBC cells by binding to the receptor for advanced glycation end products (RAGE). The functional significance of secreted Ac-APE1/Ref-1 was studied by induction of intracellular hyperacetylation through co-treatment with acetylsalicylic acid and TSA in MDA-MB-231 cells. In response to hyperacetylation, secretion of Ac-APE1/Ref-1 in vesicles was observed, resulting in significantly decreased cell viability and induction of apoptosis with increased expression of RAGE. The hyperacetylation-induced apoptosis was similar in two other TNBC cell lines: BT-459 and MDA-MB-468. Therefore, hyperacetylation may be a therapeutic target for treatment of TNBCs. This study introduces a novel paradigm whereby post-translational modification induces apoptotic cell death in breast cancer cells resistant to standard chemotherapeutic agents through secretion of auto- or paracrine molecules such as Ac-APE1/Ref-1.

Topics: Acetylation; Antigens, Neoplasm; Apoptosis; Aspirin; Breast Neoplasms; Cell Line, Tumor; Cell Membrane; Cell Survival; DNA-(Apurinic or Apyrimidinic Site) Lyase; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; MCF-7 Cells; Microscopy, Electron, Transmission; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Receptor for Advanced Glycation End Products; Triple Negative Breast Neoplasms

Breast cancer is a heterogeneous disease that can be subdivided into clinical, histopathological and molecular subtypes (luminal A-like, luminal B-like/HER2-negative, luminal B-like/HER2-positive, HER2-positive, and triple-negative). The study of new molecular factors is essential to obtain further insights into the mechanisms involved in the tumorigenesis of each tumor subtype. RASSF2 is a gene that is hypermethylated in breast cancer and whose clinical value has not been previously studied. The hypermethylation of RASSF1 and RASSF2 genes was analyzed in 198 breast tumors of different subtypes. The effect of the demethylating agent 5-aza-2'-deoxycytidine in the re-expression of these genes was examined in triple-negative (BT-549), HER2 (SK-BR-3), and luminal cells (T-47D). Different patterns of RASSF2 expression for distinct tumor subtypes were detected by immunohistochemistry. RASSF2 hypermethylation was much more frequent in luminal subtypes than in non-luminal tumors (p = 0.001). The re-expression of this gene by lentiviral transduction contributed to the differential cell proliferation and response to antineoplastic drugs observed in luminal compared with triple-negative cell lines. RASSF2 hypermethylation is associated with better prognosis in multivariate statistical analysis (P = 0.039). In conclusion, RASSF2 gene is differently methylated in luminal and non-luminal tumors and is a promising suppressor gene with clinical involvement in breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Hydroxamic Acids; Immunohistochemistry; Kaplan-Meier Estimate; Middle Aged; Multivariate Analysis; Prognosis; Proportional Hazards Models; Treatment Outcome; Tumor Suppressor Proteins

MicroRNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Increasing evidence suggests that miRNAs are functionally important in cancers. We demonstrated miR-204 exerts its function by targeting gene involved in tumor growth and chemotherapy drugs reactivity. Here, we show that Trichostatin A (TSA) could increase ERα expression in MCF-7 and MDA-MB-231 cells by reducing miR204. Analysis of tumors growth inhibition shows that TSA promotes ERα expression, which could be reversed by miR-204 mimic transfection. When miR-204 is down regulated, the inhibition of TAM on breast cancer cells is enhanced. Caspase 3 activity is also increased. TSA and TAM combination inhibits Mcl-1 expression by decreasing phosphorylation of AKT induced by ERα increase in vivo and in vitro.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line; Cell Proliferation; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; MCF-7 Cells; Mice, Nude; MicroRNAs; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-akt; Signal Transduction; Tamoxifen; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

The MAGE gene encodes cancer/testis antigens that are selectively expressed in various types of human neoplasms but not in normal tissues other than testis and placenta. However, the expression pattern of MAGE-A9 and MAGE-A11 in breast cancer patients is still unclear. The purpose of our study is to investigate the expression pattern and mechanism of MAGE-A9 and MAGE-A11 in breast cancer patients.. The expression of MAGE-A9 and MAGE-A11 was investigated in 60 breast benign diseases specimens, 60 tumor-free breast specimens and 60 breast cancer specimens by RT-PCR, and their correlation with clinicopathological parameters was elucidated. We examined the influence of the DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) together with the histone deacetylase inhibitor trichostatin A (TSA) on the expression of MAGE-A9 and MAGE-A11 genes in two breast cancer cell lines.. The expression rates of MAGE-A9 and MAGE-A11 in breast cancer specimens were 45 and 66.7%, respectively. MAGE-A9 and MAGE-A11 expression was positively associated with estrogen-receptor (ER) and HER-2 expression (p <0.05). 5-Aza-CdR treatment alone could induce the expression of MAGE-A9 and MAGE-A11 in cell lines that did not express this antigen. TSA treatment alone had no influence on MAGE-A9 and MAGE-A11 gene expression. However, TSA was able synergistically to enhance 5-aza-CdR-mediated MAGE-A transcription (p <0.05).. Our data show that MAGE-A9 and MAGE-A11 are tumor-specific antigens and not only DNA hypermethylation but also histone deacetylation is responsible for the mechanism underlying MAGE-A9 and MAGE-A11 gene silencing.

Topics: Adult; Antigens, Neoplasm; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Decitabine; DNA Modification Methylases; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Middle Aged; Neoplasm Proteins; Receptors, Estrogen

Secreted phospholipases A2 (sPLA2s) have recently been associated with several cancers, but their role in breast cancer is unknown. Here we demonstrate that mRNA expression of group IIA, III and X sPLA2s differs both in vivo in tumour biopsies and in breast cancer cells in vitro. Their expression is differentially regulated by DNA methylation and histone acetylation and, significantly, all three genes are silenced in aggressive triple negative cells due to both mechanisms. The transcription start site promoter region and the upstream CpG islands, exclusive to the group X sPLA2 gene, have variable roles in the regulation of sPLA2 expression. Our results suggest that the differential expression of hGIIA, hGIII and hGX sPLA2s in breast cancer cells is a consequence of various degrees of epigenetic silencing due to DNA hypermethylation and histone deacetylation.

Topics: Azacitidine; Breast Neoplasms; Cell Line; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Group II Phospholipases A2; Group III Phospholipases A2; Group X Phospholipases A2; Humans; Hydroxamic Acids; MCF-7 Cells; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction

COUP-TFII is reduced in endocrine-resistant breast cancer cells and is negatively associated with tumor grade. Transient re-expression of COUP-TFII restores antiestrogen sensitivity in resistant LCC2 and LCC9 cells and repression of COUP-TFII results in antiestrogen-resistance in MCF-7 endocrine-sensitive cells. We addressed the hypothesis that reduced COUP-TFII expression in endocrine-resistant breast cancer cells results from epigenetic modification. The NR2F2 gene encoding COUP-TFII includes seven CpG islands, including one in the 5' promoter and one in exon 1. Treatment of LCC2 and LCC9 endocrine-resistant breast cancer cells with 5-aza-2'-deoxycytidine (AZA), a DNA methyltransferase (DNMT) inhibitor, +/- trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, increased COUP-TFII suggesting that the decrease in COUP-TFII is mediated by epigenetic changes. Methylation-specific PCR (MSP) revealed higher methylation of NR2F2 in the first exon in LCC2 and LCC9 cells compared to MCF-7 cells and AZA reduced this methylation. Translational importance is suggested by Cancer Methylome System (CMS) analysis revealing that breast tumors have increased COUP-TFII (NR2F2) promoter and gene methylation versus normal breast.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Breast Neoplasms; COUP Transcription Factor II; CpG Islands; Decitabine; DNA Methylation; Drug Resistance, Neoplasm; Estrogen Receptor beta; Estrogen Receptor Modulators; Female; Humans; Hydroxamic Acids; MCF-7 Cells; Real-Time Polymerase Chain Reaction

Histone deacetylase inhibitors (HDACIs)-based therapies have stimulated interest via their anti-tumor activities, including apoptosis induction, cell cycle arrest, cell differentiation, and autophagy. However, the mechanisms of HDACI-associated anti-tumor activity are not yet clearly defined. The aim of this study was to explore the key events of Trichostatin A (TSA), a classic HDACI agent, against breast cancer cells.. The MCF-7, MDA-MB-231 and MCF-10A cell lines were evaluated with colony-forming and cell viability assays. Apoptosis and cell cycle distribution were detected by flow cytometry. Mitochondrial function was measured with biochemical assays, flow cytometry and transmission electron microscopy.. TSA inhibited breast cancer cell viability and proliferation, without affecting MCF-10A cell. TSA-induced breast cancer cell apoptosis was initiated by G2-M arrest and depended on mitochondrial reactive oxygen species (ROS) produced subsequent to reduced mitochondrial respiratory chain activity. The enhanced mitochondrial ROS production and apoptosis in cancer cells were markedly attenuated by antioxidants, such as N-acetyl cysteine (NAC), reduced glutathione (GSH) and Vitamin C.. The present study demonstrated that TSA-induced cell death by arresting cell cycle in G2-M phase and was dependent on production of mitochondria-derived ROS, which was derived from impaired mitochondrial respiratory chain.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Proliferation; Cytochromes c; Electron Transport; Female; G2 Phase; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MCF-7 Cells; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species

ARHI is a maternally imprinted tumor suppressor gene that is expressed in normal breast epithelial cells but not in most breast cancer cells. Aberrant methylation and hypernomic histone deacetylation have been implicated in the silencing of ARHI. To investigate the mechanism of ARHI induction, MDA-MB-231 breast cancer cells were either transfected with the eukaryotic expression vector, pcDNA3.1(+)-ARHI, or were simultaneously treated with a histone deacetylase inhibitor, [trichostatin A, (TSA)] and the methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC). The latter treatment group also included the targeting of ARHI by small interfering RNA (siRNA) to further examine interactions between ARHI and the drugs applied. Levels of ARHI were detected by western blotting, MTT assays were used to evaluate cell proliferation, and both cell cycle progression and apoptosis were detected using flow cytometry. Both the transfection of pcDNA3.1(+)‑ARHI and the application of TSA+DAC induced the expression of ARHI. Furthermore, reduced cell proliferation, cell cycle arrest and enhanced apoptosis were observed for both groups compared to controls. However, a G1/S cell cycle arrest was observed for the pcDNA3.1(+)-ARHI group, while a G2 cell cycle arrest was observed for the TSA+DAC group. The latter effect was reversed with the introduction of ARHI-targeted siRNA in combination with TSA+DAC treatment. To further clarify these observations, expression levels of several key cell cycle regulators were analyzed by western blotting. The pcDNA3.1(+)-ARHI group exhibited higher expression levels of p53, p21 and p27, and lower levels of cyclin D1, CDK4 and CDK6 when compared to the control group (P<0.05). For the TSA+DAC group, higher levels of p53, p21, cyclin B1 and Chk1 were detected, concomitant with lower levels of CDK1, when compared to the control group. Taken together, these results suggest that ARHI acts as a tumor suppressor gene in MDA-MB-231 cells and, although TSA+DAC can block the cells at different cell cycle phage, the antitumor effect is ARHI-dependent.

Topics: Apoptosis; Azacitidine; Breast Neoplasms; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Decitabine; Female; G2 Phase Cell Cycle Checkpoints; Genetic Vectors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Methyltransferases; Protein Kinases; rho GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; S Phase Cell Cycle Checkpoints; Transfection; Tumor Suppressor Protein p53

Mammalian telomeric DNA consists of tandem repeats of the sequence TTAGGG associated with a specialized set of proteins, known collectively as Shelterin. These telosomal proteins protect the ends of chromosomes against end-to-end fusion and degradation. Short telomeres in breast cancer cells confer telomere dysfunction and this can be related to Shelterin proteins and their level of expression in breast cancer cell lines. This study investigates whether expression of Shelterin and Shelterin-associated proteins are altered, and influence the protection and maintenance of telomeres, in breast cancer cells. 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A (TSA) were used in an attempt to reactivate the expression of silenced genes. Our studies have shown that Shelterin and Shelterin-associated genes were down-regulated in breast cancer cell lines; this may be due to epigenetic modification of DNA as the promoter region of POT1 was found to be partially methylated. Shelterin genes expression was up-regulated upon treatment of 21NT breast cancer cells with 5-aza-CdR and TSA. The telomere length of treated 21NT cells was measured by q-PCR showed an increase in telomere length at different time points. Our studies have shown that down-regulation of Shelterin genes is partially due to methylation in some epithelial breast cancer cell lines. Removal of epigenetic silencing results in up-regulation of Shelterin and Shelterin-associated genes which can then lead to telomere length elongation and stability.

Topics: Antineoplastic Agents; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Decitabine; DNA; DNA Methylation; Epigenesis, Genetic; Female; Histones; Humans; Hydroxamic Acids; MCF-7 Cells; Methyltransferases; Promoter Regions, Genetic; Shelterin Complex; Telomere; Telomere Homeostasis; Telomere-Binding Proteins

Partial loss of large ribosomal subunit protein 24 (RPL24) function is known to protect mice against Akt or Myc-driven cancers, in part via translational inhibition of a subset of cap(eIF4E)-dependently translated mRNAs. The role of RPL24 in human malignancies is unknown. By analyzing a public dataset of matched human breast cancers and normal mammary tissue, we found that breast cancers express significantly more RPL24 than matched normal breast samples. Depletion of RPL24 in breast cancer cells by >70% reduced cell viability by 80% and decreased protein expression of the eIF4E-dependently translated proteins cyclin D1 (75%), survivin (46%) and NBS1 (30%) without altering GAPDH or beta-tubulin levels. RPL24 knockdown also reduced 80S subunit levels relative to 40S and 60S levels. These effects on expression of eIF4E-dependent proteins and ribosome assembly were mimicked by 2-24 h treatment with the pan-HDACi, trichostatin A (TSA), which induced acetylation of 15 different polysome-associated proteins including RPL24. Furthermore, HDAC6-selective inhibition or HDAC6 knockdown induced ribosomal protein acetylation. Via mass spectrometry, we found that 60S-associated, but not, polysome-associated, RPL24 undergoes HDACi-induced acetylation on K27. Thus, RPL24 K27 acetylation may play a role in ribosome assembly. These findings point toward a novel acetylation-dependent polysome assembly mechanism regulating tumorigenesis.

Topics: Acetylation; Amino Acid Sequence; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Eukaryotic Initiation Factors; Gene Expression Regulation, Neoplastic; HEK293 Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lysine; Mass Spectrometry; Molecular Sequence Data; Polyribosomes; Ribosomal Proteins; RNA Interference

The Library of Integrated Network-Based Cellular Signatures (LINCS) project aims to create a network-based understanding of biology by cataloging changes in gene expression and signal transduction that occur when cells are exposed to a variety of perturbations. It is helpful for understanding cell pathways and facilitating drug discovery. Here, we developed a novel approach to infer cell-specific pathways and identify a compound's effects using gene expression and phosphoproteomics data under treatments with different compounds. Gene expression data were employed to infer potential targets of compounds and create a generic pathway map. Binary linear programming (BLP) was then developed to optimize the generic pathway topology based on the mid-stage signaling response of phosphorylation. To demonstrate effectiveness of this approach, we built a generic pathway map for the MCF7 breast cancer cell line and inferred the cell-specific pathways by BLP. The first group of 11 compounds was utilized to optimize the generic pathways, and then 4 compounds were used to identify effects based on the inferred cell-specific pathways. Cross-validation indicated that the cell-specific pathways reliably predicted a compound's effects. Finally, we applied BLP to re-optimize the cell-specific pathways to predict the effects of 4 compounds (trichostatin A, MS-275, staurosporine, and digoxigenin) according to compound-induced topological alterations. Trichostatin A and MS-275 (both HDAC inhibitors) inhibited the downstream pathway of HDAC1 and caused cell growth arrest via activation of p53 and p21; the effects of digoxigenin were totally opposite. Staurosporine blocked the cell cycle via p53 and p21, but also promoted cell growth via activated HDAC1 and its downstream pathway. Our approach was also applied to the PC3 prostate cancer cell line, and the cross-validation analysis showed very good accuracy in predicting effects of 4 compounds. In summary, our computational model can be used to elucidate potential mechanisms of a compound's efficacy.

Topics: Antineoplastic Agents; Benzamides; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Digoxigenin; Female; Gene Expression; Gene Regulatory Networks; Genomics; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; MCF-7 Cells; Phosphorylation; Programming, Linear; Proteome; Proteomics; Pyridines; Signal Transduction; Staurosporine; Tumor Suppressor Protein p53

Histone deacetylases (HDACs) are important in chromatin remodeling and epigenetic regulation of gene expression. Histone deacetylase inhibitors (HDACi) have highly effective anti-metastatic and anti-angiogenic activity in various types of cancer, while the molecular mechanisms involved in this process are not fully understood. In the present study, trichostatin A (TSA), a HDACi, was found to suppress MCF-7 breast carcinoma cell invasion and upregulate TET1 expression in a dose-dependent manner. TET1, a dioxygenase involved in cytosine demethylation, is downregulated during breast cancer progression. TET1 knockdown in MCF-7 cells facilitates cell invasion, inhibits the expression of tissue inhibitors of metalloproteinase 2/3 (TIMP2/3) and promotes matrix metalloproteinases (MMP) 2/9 transcriptional activity. Importantly, TET1 depletion impaired the inhibitory effect of TSA on breast cancer cell invasion. Together, these results illustrated a mechanism by which TET1 partially mediates HDACi elicited suppression of breast cancer invasion.

Topics: Adult; Antineoplastic Agents; Breast Neoplasms; Cell Movement; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; MCF-7 Cells; Middle Aged; Mixed Function Oxygenases; Neoplasm Invasiveness; Proto-Oncogene Proteins; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinase-3

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl from lysine residues in histones and other proteins, which results in gene transcriptional repression and subsequent changes in signaling events. HDACs inhibitors (HDACIs) have been used to reverse the aberrant epigenetic changes associated with cancer. However, the effects of HDACIs on epithelial-mesenchymal transition (EMT) in human cancer cells remain unclear. EMT is a fundamental process governing morphogenesis in multicellular organisms and promotes cancer invasion and metastasis. In this study, human cancer cells were treated with the HDACI trichostatin A (TSA). TSA was found to induce mesenchymal‑like morphological changes in BGC-823 human gastric cancer and MCF-7 breast cancer cells, and increase the expression levels of the mesenchymal markers Vimentin and Twist. However, the expression levels of the epithelial cell marker E-cadherin were also increased in response to TSA treatment, while cell migration was reduced by TSA. Furthermore, TSA decreased cancer cell colony formation in BGC-823 and MCF-7 cells, and led to the deregulation of β-catenin, a critical signaling molecule involved in EMT. In conclusion, the results suggested that TSA exhibits dual functions in EMT induction and inhibition in human cancer cells, but the detailed mechanisms require further investigation.

Topics: beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Gene Expression; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; MCF-7 Cells; Nuclear Proteins; Signal Transduction; Twist-Related Protein 1; Vimentin

Treatment with histone deacetylase inhibitors (HDACI) results in potent cytotoxicity of a variety of cancer cell types, and these drugs are used clinically to treat hematological tumors. They are known to repress the transcription of ERBB2 and many other oncogenes, but little is known about this mechanism. Using global run-on sequencing (GRO-seq) to measure nascent transcription, we find that HDACI cause transcriptional repression by blocking RNA polymerase II elongation. Our data show that HDACI preferentially repress the transcription of highly expressed genes as well as high copy number genes in HER2+ breast cancer genomes. In contrast, genes that are activated by HDACI are moderately expressed. We analyzed gene copy number in combination with microarray and GRO-seq analysis of expression level, in normal and breast cancer cells to show that high copy number genes are more likely to be repressed by HDACI than non-amplified genes. The inhibition of transcription of amplified oncogenes, which promote survival and proliferation of cancer cells, might explain the cancer-specific lethality of HDACI, and may represent a general therapeutic strategy for cancer.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Female; Gene Dosage; Gene Expression Regulation, Neoplastic; Gene Silencing; Genome, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Oligonucleotide Array Sequence Analysis; Oncogenes; Receptor, ErbB-2; RNA Polymerase II; RNA, Messenger; Transcription Elongation, Genetic; Transcription Initiation, Genetic; Transcriptome

Heterogeneity of cellular phenotypes in asynchronous cell populations placed in the same biochemical and biophysical environment may depend on cell cycle and chromatin modifications; however, no current method can measure these properties at single-cell resolution simultaneously and in situ. Here, we develop, test, and validate a new microscopy assay that rapidly quantifies global acetylation on histone H3 and measures a wide range of cell and nuclear properties, including cell and nuclear morphology descriptors, cell-cycle phase, and F-actin content of thousands of cells simultaneously, without cell detachment from their substrate, at single-cell resolution. These measurements show that isogenic, isotypic cells of identical DNA content and the same cell-cycle phase can still display large variations in H3 acetylation and that these variations predict specific phenotypic variations, in particular, nuclear size and actin cytoskeleton content, but not cell shape. The dependence of cell and nuclear properties on cell-cycle phase is assessed without artifact-prone cell synchronization. To further demonstrate its versatility, this assay is used to quantify the complex interplay among cell cycle, epigenetic modifications, and phenotypic variations following pharmacological treatments affecting DNA integrity, cell cycle, and inhibiting chromatin-modifying enzymes.

Topics: Acetylation; Actins; Animals; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Shape; Chromatin; Culture Media, Serum-Free; DNA; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Mice; Microscopy, Fluorescence; Myoblasts; Reproducibility of Results; Single-Cell Analysis

Here, we report a complex regulation of endothelin-1 (ET-1) axis driven by epigenetic reactions in 1833-bone metastatic cells, emphasizing the importance in skeletal metastasis from breast carcinoma. Inhibitors of histone deacetylases, trichostatin A (TSA), and of DNA methylases, 5'-Azacytidine (Aza), caused, respectively, reduction and increase in 1833 cell invasiveness, without affecting the basal migration of parental MDA-MB231 cells. Of note, in the two cell lines exposed to Aza the blockade of the ET-1 receptor ETAR with BQ-123 oppositely changed invasive properties. Even if in MDA-MB231 cells the ET-1 axis was scarcely influenced by epigenetic reactions, ETAR remarkably decreased after Aza. In contrast, in 1833 cells Aza exposure enhanced ET-1 coupled to ETAR wild type, being also ETAR truncated form increased, and invasiveness was stimulated. Under demethylation, the increase in ET-1 steady state protein level in 1833 clone seemed regulated at transcriptional level principally via Ets1 transcription factor. In fact, actinomycin D almost completely prevented ET-1 mRNA induction due to Aza. Only in 1833 cells, TSA exposure inactivated ET-1 axis, with reduction of the expression of ET-1 and ETAR mutated form, in agreement with Matrigel invasion decrease. This treatment favoured the ET-1 repressional control, taking place at the level of mRNA stability due to the 3'-untranslated region in the ET-1 gene, and also decreased transcription via NF-kB. Environmental conditions that alter the balance between epigenetic reactions might, therefore, affect metastasis migratory mode influencing ET-1 axis.

Topics: Azacitidine; Bone Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Collagen; Dactinomycin; DNA Methylation; Drug Combinations; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Laminin; Mutation; Neoplasm Invasiveness; Peptides, Cyclic; Proteoglycans; Proto-Oncogene Protein c-ets-1; Receptor, Endothelin A; RNA Stability; RNA, Messenger; Transcription, Genetic

The purpose of this work is to determine the molecular mechanisms underlying tamoxifen resistance. We show here that ER-β is epigenetically silenced in a cell line with acquired tamoxifen resistance (MCF-7/TAM-R) and this could be reversed by 5-AZA-deoxycytidine (5-AZA) and trichostatin-A (TSA) pre-treatment. Subsequent treatment with 4-hydroxy-tamoxifen (4-OHT) induced ER-β nuclear translocation, upregulated pS2 and p21 levels and reduced cell viability. Transfection with an ER-β expression vector sensitized MCF-7/TAM-R cells to the growth inhibitory and pro-apoptotic effects of 4-OHT, indicating that ER-β re-expression alone is sufficient to restore sensitivity to tamoxifen. This novel finding reveals that ER-β is fundamental in overcoming acquired tamoxifen resistance and provides insights for new therapeutic protocols against breast cancer.

Topics: Active Transport, Cell Nucleus; Antineoplastic Agents, Hormonal; Apoptosis; Azacitidine; Breast Neoplasms; Cell Survival; Chromatin Assembly and Disassembly; Decitabine; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MCF-7 Cells; Tamoxifen; Time Factors; Transfection

Expression of claudin-1, a tight junction protein, is highly upregulated in colon cancer. We have reported that claudin-1 expression in colon cancer cells is epigenetically regulated as histone deacetylase (HDAC) inhibitors decrease claudin-1 messenger RNA (mRNA) stability and thus expression. In this regard, our data suggested a role of the 3'-untranslated region (UTR) in the regulation of HDAC-dependent regulation of claudin-1 mRNA stability. In the current study, we demonstrate, based on our continued investigation, that the ELAV-like RNA-binding proteins (RBPs), human antigen R (HuR) and tristetraprolin (TTP) associate with the 3'-UTR of claudin-1 mRNA to modulate the latter's stability. Ribonomic and site-directed mutagenesis approaches were used to confirm the binding of HuR and TTP to the 3'-UTR of claudin-1. We further confirmed their roles in the stabilization of claudin-1 mRNA, under conditions of HDAC inhibition. In summary, we report that HuR and TTP are the critical regulators of the posttranscriptional regulation of claudin-1 expression in colon cancer cells. We also demonstrate that inhibition of HDACs by trichostatin treatment decreased the binding of HuR while increasing the binding of TTP to the 3'-UTR of claudin-1. Additionally, we provide data showing transcriptional regulation of claudin-1 expression, through the regulation of transcription factor Sp1. Taken together, we demonstrate epigenetic regulation of claudin-1 expression in colon cancer cells at the transcriptional and posttranscriptional levels.

Topics: 3' Untranslated Regions; Base Sequence; Blotting, Western; Breast Neoplasms; Cells, Cultured; Chromatin Immunoprecipitation; Claudin-1; Colonic Neoplasms; ELAV Proteins; Epigenesis, Genetic; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Kidney; Molecular Sequence Data; Mutagenesis, Site-Directed; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tristetraprolin

ERβ1 is often down-regulated in breast cancer compared to normal breast but mechanisms surrounding this are unclear. We examined whether loss of heterozygosity (LOH) or methylation at ERβ promoters (0N, 0K) and/or untranslated exon 0N were involved in ERβ down-regulation in breast cancer tissues and cell lines and if treatment with the de-methylating agent 5-aza-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A could influence expression in vitro. We found no evidence of correlation between LOH at 14q22-24 (genomic locus containing ERβ/ESR2), and ERβ1 expression in primary breast cancers. A negative correlation between ERβ1 mRNA expression and methylation status was observed for promoter 0N in BT-20, MDA-MB-453 and T47D cells. Promoter 0K was consistently unmethylated. In primary breast tumours, methylation of the untranslated exon 0N, downstream of promoter 0N, but not of promoter 0N itself, correlated with down-regulation of ERβ. In MDA-MB-453 cells, treatment with 5-aza-deoxycytidine was sufficient to induce ERβ1 expression from the 0N promoter while in BT-20 both agents were required. Examination of various sites on ESR2 highlighted epigenetic but not genetic regulation of ERβ1. In particular methylation adjacent to promoter 0N was a key regulatory event for ERβ1 silencing. A combination of de-methylating agents and histone deacetylase inhibitors fully restored ERβ1 expression which may offer a novel therapeutic angle for breast cancer management.

Topics: 5' Untranslated Regions; Acetylation; Azacitidine; Breast Neoplasms; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Down-Regulation; Enzyme Inhibitors; Epigenesis, Genetic; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Loss of Heterozygosity; Promoter Regions, Genetic; RNA, Messenger

To investigate the effect of tricostantin A (TSA) on self-renewal of breast cancer stem cells and explore the mechanisms.. Breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 and SKBR3 were cultured in suspension and treated with different concentrations of TSA for 7 days, using 0.1% DMSO as the control. Secondary mammosphere formation efficiency and percentage of CD44(+)/CD24(-) sub-population in the primary mammospheres were used to evaluate the effects of TSA on self-renewal of breast cancer stem cells. The breast cancer stem cell surface marker CD44(+)/CD24(-) and the percentage of apoptosis in the primary mammospheres were assayed using flow cytometry. The mRNA expressions of Nanog, Sox2 and Oct4 in the primary mammospheres were assayed with quantitative PCR.. TSA at both 100 and 500 nmol/L, but not at 10 nmol/L, partially inhibited the self-renewal of breast cancer stem cells from the 4 cell lines. TSA at 500 nmol/L induced cell apoptosis in the primary mammospheres. TSA down-regulated the mRNA expression of Nanog and Sox2 in the primary mammospheres.. TSA can partially inhibit the self-renewal of breast cancer stem cells through a mechanism involving the down-regulation of Nanog and Sox2 expression, indicating the value of combined treatments with low-dose TSA and other anticancer drugs to achieve maximum inhibition of breast cancer stem cell self-renewal. The core transcriptional factor of embryonic stem cells Nanog and Sox2 can be potential targets of anticancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; CD24 Antigen; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Female; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hyaluronan Receptors; Hydroxamic Acids; Nanog Homeobox Protein; Neoplastic Stem Cells; RNA, Messenger; SOXB1 Transcription Factors

MTO1 and MRPL41 are nuclear-encoded mitochondrial genes encoding a mitochondrial tRNA-modifying enzyme and a mitochondrial ribosomal protein, respectively. Although both genes have been known to have potential roles in cancer, little is known about their molecular regulatory mechanism, particularly from an epigenetic approach. In this study, we aimed to address their epigenetic regulation through the estrogen receptor (ER) in breast cancer.. Digital differential display (DDD) was conducted to identify mammary gland-specific gene candidates including MTO1 and MRPL41. Promoter CpG methylation and expression in breast cancer cell lines and tissues were examined by methylation-specific PCR and real time RT-PCR. Effect of estradiol (E2), tamoxifen, and trichostatin A (TSA) on gene expression was examined in ER + and ER- breast cancer cell lines. Chromatin immunoprecipitation and luciferase reporter assay were performed to identify binding and influencing of the ER to the promoters.. Examination of both cancer tissues and cell lines revealed that the two genes showed an opposite expression pattern according to ER status; higher expression of MTO1 and MRPL41 in ER- and ER+ cancer types, respectively, and their expression levels were inversely correlated with promoter methylation. Tamoxifen, E2, and TSA upregulated MTO1 expression only in ER+ cells with no significant changes in ER- cells. However, these chemicals upregulated MRPL41 expression only in ER- cells without significant changes in ER+ cells, except for tamoxifen that induced downregulation. Chromatin immunoprecipitation and luciferase reporter assay identified binding and influencing of the ER to the promoters and the binding profiles were differentially regulated in ER+ and ER- cells.. These results indicate that different epigenetic status including promoter methylation and different responses through the ER are involved in the differential expression of MTO1 and MRPL41 in breast cancer.

Topics: Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; DNA Methylation; Epigenesis, Genetic; Estradiol; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Mitochondrial Proteins; Promoter Regions, Genetic; Protein Binding; Receptors, Estrogen; Response Elements; Ribosomal Proteins; RNA-Binding Proteins; Tamoxifen

Breast cancer patients with HER-2 positive or estrogen receptor negative tumors have a poor prognosis because these tumors are aggressive and respond poorly to standard therapies. Histone deacetylase (HDAC) inhibitors have been shown to decreased cell survival, which suggests that HDAC inhibitors may be developed for preventing and treating breast cancer. Curcumin has anti-inflammatory and proapoptotic effects in cancer cells. We determined whether the HDAC inhibitor, Tricostatin A (TSA) in combination with curcumin would produce greater antiproliferative and apoptotic effects than either agent alone. Increasing the concentration of curcumin from 10 to 20 µM enhanced the growth inhibitory effects of the combination in SkBr3 and 435eB breast cancer cells, which was accompanied by decreased viability along with decreased phosphorylation of ERK and Akt. The decreased cell viability observed in SkBr3 cells when curcumin was combined with TSA led to a G0/G1 cell cycle arrest and increased p21 and p27, and decreased Cyclin D1 protein expression. The combination induced cleavage of caspase 3 and poly(ADP-ribose) polymerase-1, suggesting that cell death occurred by apoptosis. There were no changes in protein expression of Bcl2, Bax, or Bcl-xL and decreased expression of p53. The combination increased protein expression of phosphorylated JNK and phosphorylated p38. Pharmacological inhibition of JNK, but not p38, attenuated the decreased viability induced by the curcumin and TSA combination. We conclude that p53 independent apoptosis induced by combining curcumin and TSA involves JNK activation. These findings provide a rationale for exploring the potential benefits of the combination of curcumin with TSA for treatment of breast cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Curcumin; Cyclin D1; Enzyme Inhibitors; Female; Humans; Hydroxamic Acids; Imidazoles; MAP Kinase Kinase 4; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyridines

15-Lipoxygenase-1 (15-Lox-1) is a key enzyme mediating oxidative metabolism of polyunsaturated fatty acids and has attracted considerable interest as a potential target for the induction of apoptosis in cancer cells. Knowledge of relationship between 15-Lox-1 and histone deacetylase inhibitors is lacking in the breast cancer. This study is aimed to investigate the role of Trichostatin A (TSA) and 13(S)-HODE, as a metabolite of 15-Lox-1, in the regulation of breast cancer cell growth. The cytotoxic effect of TSA, as a potent HDAC inhibitor, was measured using MTT assay. Annexin V-FITC and PI staining were performed to detect apoptosis and cell cycle distribution using Flow cytometry. The role of 15-Lox-1 in the regulation of cell growth was assessed by 15-Lox-1 inhibitor and the level of 15-Lox-1 metabolite was measured to determine 15-Lox activity after treatment by TSA. The results demonstrated that TSA induced cell growth inhibition via 15-Lox-1, in a dose- and time-dependent manner, and subsequently accompanied by the cell cycle arrest and induction of apoptosis. Moreover, growth inhibitory effect of TSA was associated with the elevation of 15-Lox-1 metabolite (13(S)-HODE). This study provided evidences that the inhibitory effect of TSA on the breast cancer cell growth occurs via the induction of 15-Lox-1 activity and 13(S)-HODE production. Our findings underline the possible role of 15-Lox-1/13(S)-HODE pathway as a promising molecular approach for the induction of apoptosis in breast cancer cells.

Topics: Apoptosis; Arachidonate 15-Lipoxygenase; Breast Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Linoleic Acids; Lipoxygenase Inhibitors

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1, and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer.

Topics: Azacitidine; Binding Sites; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Embryonal; Cell Movement; Decitabine; DNA Methylation; DNA Modification Methylases; Dose-Response Relationship, Drug; Embryonal Carcinoma Stem Cells; Female; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genes, Reporter; GPI-Linked Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Luciferases; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Proteins; Nuclear Receptor Subfamily 6, Group A, Member 1; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; RNA Interference; RNA, Messenger; Time Factors; Tissue Array Analysis; Transcription, Genetic; Transfection; Tretinoin; Valproic Acid

Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and normally does not respond to conventional estrogen target-directed therapies. The soybean isoflavone, genistein (GE), has been shown to prevent and inhibit breast cancer and recent studies have suggested that GE can enhance the anticancer capacity of an estrogen antagonist, tamoxifen (TAM), especially in ERα-positive breast cancer cells. However, the role of GE in ERα-negative breast cancer remains unknown.. We have evaluated the in vitro and in vivo epigenetic effects of GE on ERα reactivation by using MTT assay, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay, western-blot assay, immunoprecipitation (ChIP) assay, immunohistochemistry and epigenetic enzymatic activity analysis. Preclinical mouse models including xenograft and spontaneous breast cancer mouse models were used to test the efficacy of GE in vivo.. We found that GE can reactivate ERα expression and this effect was synergistically enhanced when combined with a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), in ERα-negative MDA-MB-231 breast cancer cells. GE treatment also re-sensitized ERα-dependent cellular responses to activator 17β-estradiol (E2) and antagonist TAM. Further studies revealed that GE can lead to remodeling of the chromatin structure in the ERα promoter thereby contributing to ERα reactivation. Consistently, dietary GE significantly prevented cancer development and reduced the growth of ERα-negative mouse breast tumors. Dietary GE further enhanced TAM-induced anti-cancer efficacy due at least in part to epigenetic ERα reactivation.. Our studies suggest that soybean genistein can epigenetically restore ERα expression, which in turn increases TAM-dependent anti-estrogen therapeutic sensitivity in vitro and in vivo. The results from our studies reveal a novel therapeutic combination approach using bioactive soybean product and anti-hormone therapy in refractory ERα-negative breast cancer which will provide more effective options in breast cancer therapy.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents, Hormonal; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Drug Resistance, Neoplasm; Drug Synergism; Epigenesis, Genetic; Estradiol; Estrogen Receptor alpha; Female; Gene Expression; Genistein; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Mice; Mice, Nude; Mice, Transgenic; Proliferating Cell Nuclear Antigen; Promoter Regions, Genetic; Tamoxifen; Tumor Burden; Xenograft Model Antitumor Assays

The development of drug resistance represents a major complication in the effective treatment of breast cancer. Epigenetic therapy, through the use of histone deacetylase inhibitors (HDACi) or demethylation agents, is an emerging area of therapeutic targeting in a number of ontological entities, particularly in the setting of aggressive therapy-resistant disease. Using the well-described HDAC inhibitor trichostatin A (TSA) we demonstrate the suppression of in vitro clonogenicity in the previously described apoptosis-resistant MCF-7TN-R breast carcinoma cell line. Additionally, recent work has demonstrated that these agents can alter the expression profile of microRNA signatures in malignant cells. Using an unbiased microRNA microarray analysis, changes in miRNA expression of MCF-7TN-R cells treated with TSA for 24 h were analyzed. We observed significant up-regulation of 22 miRNAs and down-regulation of 10 miRNAs in response to TSA treatment. Our results demonstrate that the HDACi, TSA, exerts anticancer activity in the apoptosis-resistant MCF-7TN-R breast carcinoma cell line. This activity is correlated with TSA alteration of microRNA expression profiles indicative of a less aggressive phenotype.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Gene Expression; Gene Expression Profiling; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; MicroRNAs; Oligonucleotide Array Sequence Analysis

It has recently been demonstrated that phospholipase C gamma-1 (PLCγ1) activation may contribute to breast carcinoma cell motility and their metastasis. Employing MCF-7 breast cancer cells, we showed the effect of trichostatin A (TSA) on the cellular contents of the PLCγ1 molecule. Using reverse transcription, real-time quantitative PCR and western blot analysis, we demonstrated that TSA reduced the PLCγ1 transcript and protein levels in MCF-7 cells. We also found that TSA decreased the half-life of the PLCγ1 transcript from approximately 7hours to 5hours. Moreover, we observed that protein synthesis appears to be essential in the TSA reduction of PLCγ1 mRNA stability. Since PLCγ1 activation is considered a key factor in the initiation of events that increase malignant cell motility, our observations may support the validity of TSA in anticancer studies.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Half-Life; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Phospholipase C gamma; Protein Synthesis Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Antiestrogen is one type of the endocrine therapeutic agents for estrogen receptor α (ERα)-positive breast cancer. Unfortunately, this treatment alone is insufficient. Here we reported a novel potential anticancer strategy by using histone deacetylase (HDAC) inhibitor to enhance the action of endocrine therapy in ERα-positive breast cancer cell. The well-described HDAC inhibitor, trichostatin A (TSA), and antiestrogen raloxifene were found to, respectively, inhibit E2-induced proliferation of MCF-7 breast cancer cell in a dose-responsive and time-dependent manner. TSA and raloxifene enhanced the antiproliferative activity of each other by promoting cell death via apoptosis and cell cycle arrest. Thus, they displayed better antiproliferative effects in combined treatment than that with either agent alone. The expression level of estrogen receptor β (ERβ) showed a marked increase after TSA or/and raloxifene treatment. Treatments with TSA or/and raloxifene resulting in the up-regulation of ERβ are in accordance with the antiproliferative effects of the two agents. Furthermore, the over-expression of ERβ by adenovirus delivery could inhibit the proliferation of MCF-7 tumor cells and drastically enhanced the antiproliferative effects of TSA and raloxifene. These results demonstrated that the interference of ERβ on the antiproliferative effects of HDAC inhibitor and antiestrogen constitutes a promising approach for breast cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Cyclin B; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Drug Synergism; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Inhibitory Concentration 50; Raloxifene Hydrochloride; Receptors, Progesterone; Trefoil Factor-1; Tumor Suppressor Proteins

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/β-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/β-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/β-catenin mRNA levels and activated TCF4/β-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/β-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and β-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/β-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/β-catenin complex activity.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Breast Neoplasms; Carbohydrate Epimerases; Chromatin; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; MCF-7 Cells; Promoter Regions, Genetic; Protein Processing, Post-Translational; Transcription Factor 4; Transcription Factors; Transcription, Genetic; Wnt Signaling Pathway

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.

Topics: Adipose Tissue; Azacitidine; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Receptors, Prostaglandin E, EP2 Subtype; Reference Values; Stromal Cells

We have previously reported that although SOX9 is a transcription factor, it is often localized in the cytoplasm of some invasive and metastatic breast carcinomas. To determine whether cytoplasmic compartmentalization is a common mechanism utilized by cancer cells to proliferate indefinitely, SOX9 localization was examined at different stages of development in normal mouse mammary glands and in cancer cells. We show here that SOX9 expression is nuclear in ductal epithelial cells throughout mammary gland development and differentiation but is localized in the cytoplasm of some breast cancer cell lines (BCCLs). Furthermore, cytoplasmic localization of SOX9 is associated with abrogation of the growth arrest response of breast cancer cells and results from dysregulated HDAC activity rather than defects in its nuclear export. Immuno-precipitation and immunoblot studies revealed that inhibiting HDAC activity with Trichostatin A can rescue this defect by up-regulating acetylated SOX9 and p21 expression that results in increased cell death. Our data suggests nuclear SOX9 expression parallels development and differentiation but cytoplasmic SOX9 expression is associated with abrogation of growth arrest response of breast cancer cells. Such expressions may be a common mechanism utilized by some transformed breast cancer cells to proliferate indefinitely.

Topics: Animals; Breast Neoplasms; Cell Growth Processes; Cell Line, Tumor; Cell Nucleus; Cytoplasm; Fatty Acids, Unsaturated; Female; Gene Expression; Humans; Hydroxamic Acids; Immunohistochemistry; Mammary Glands, Animal; Mice; Pregnancy; SOX9 Transcription Factor; Transfection

The LHR has an essential role in sexual development and reproductive function, and its transcription is subjected to several modes of regulation. In this study, we investigated PC4 coactivator function in the control of LHR transcription. Knockdown of PC4 by siRNA inhibited the LHR basal promoter activity and trichostatin A (TSA)-induced gene transcriptional activation and expression in MCF-7 cells. While overexpression of PC4 alone had no effect on the LHR gene, it significantly enhanced Sp1- but not Sp3-mediated LHR transcriptional activity. PC4 directly interacts with Sp1 at the LHR promoter, and this interaction is negatively regulated by PC4 phosphorylation. The coactivator domain (22-91 aa) of PC4 and DNA binding domain of Sp1 are essential for PC4/Sp1 interaction. ChIP assay revealed significant occupancy of PC4 at the LHR promoter that increased upon TSA treatment. Disruption of PC4 expression significantly reduced TSA-induced recruitment of TFIIB and RNAP II, at the promoter. PC4 functions are beyond TSA-induced phosphatase release, PI3K-mediated Sp1 phosphorylation, and HDAC1/2/mSin3A co-repressor release indicating its role as linker coactivator of Sp1 and the transcriptional machinery. These findings demonstrated a critical aspect of LHR modulation whereby PC4 acts as a coactivator of Sp1 to contribute to the human of LHR transcription.

Topics: Breast Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Female; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Receptors, LH; RNA Polymerase II; RNA, Small Interfering; Sp1 Transcription Factor; Transcription Factor TFIIB; Transcription Factors; Transcription, Genetic

ARHI is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma in situ (DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel.. Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts.. ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest.. ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Azacitidine; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Decitabine; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Hydroxamic Acids; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Paclitaxel; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA Interference; Transplantation, Heterologous

Aberrant DNA methylation is deeply involved in the development and progression of human breast cancers, but its inducers and molecular mechanisms are still unclear. To reveal such inducers and clarify the molecular mechanisms, animal models are indispensable. Here, to identify genes silenced by promoter DNA methylation in rat mammary carcinomas, we took a combined approach of methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray analysis and expression microarray analysis after treatment with epigenetic drugs. MeDIP-CGI microarray revealed that among 5031 genes with promoter CGI, 465 were methylated in a carcinoma cell line induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), but not in normal mammary epithelial cells. By treatment of the cell line with 5-aza-2'-deoxycytidine and trichostatin A, 29 of the 465 genes were shown to be re-expressed. In primary mammary carcinomas, five (Angptl4, Coro1a, RGD1304982, Tmem37 and Ndn) of the 29 genes were methylated in one or more of 25 samples. Quantitative expression analysis revealed that Angptl4 had high expression in normal mammary glands, but low expression in primary carcinomas. Also in humans, ANGPTL4 was unmethylated and expressed in normal mammary epithelial cells, but was methylated in 11 of 91 (12%) primary breast cancers. This is the first study to identify genes aberrantly methylated in rat mammary carcinomas, and Angptl4 is a novel methylation-silenced gene both in rat and human mammary carcinomas. The combination of the MeDIP-CGI microarray analysis and expression microarray analysis after treatment with epigenetic drugs was effective in reducing the number of methylated genes that are not methylation silenced.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Animals; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Decitabine; DNA Methylation; Female; Gene Silencing; Hydroxamic Acids; Mammary Glands, Animal; Mammary Neoplasms, Animal; Oligonucleotide Array Sequence Analysis; Rats; Rats, Inbred F344; Rats, Sprague-Dawley

Thymidine phosphorylase (TP) is an enzyme involved in thymidine synthesis and degradation. The expression of this enzyme has been proposed as a predictive factor for the therapeutic benefit of capecitabine, which is a precursor of the drug 5'-fluorouracil. In fact, TP is the rate-limiting enzyme in the activation of capecitabine. Therefore, higher levels of TP are expected to sensitize cancer cells to capecitabine treatment. In the present study, using breast cancer cell lines, we found a correlation between TP mRNA and protein levels, suggesting that compounds able to increase TP gene expression also increase protein levels. Accordingly, we demonstrated that treatment of breast cancer MCF7 and MDA231 cell lines with histone deacetylase inhibitors, tricostatin A and suberoylanilide hydroxamic acid, increased TP both at the mRNA and protein level. The effects of histone deacetylase inhibitors were not found to occur via the cytokine TNFα, a known inducer of TP expression. Our findings suggest a strategy to sensitize breast cancer cells to capecitabine treatment.

Topics: Breast Neoplasms; Capecitabine; Cell Line, Tumor; Cell Survival; Deoxycytidine; Drug Synergism; Female; Fluorouracil; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thymidine Phosphorylase; Vorinostat

Thyroid hormones regulate cell proliferation, differentiation as well as apoptosis. However molecular mechanism underlying apoptosis as a result of thyroid hormone signaling is poorly understood. The antiapoptotic role of Senescence Marker Protein-30 (SMP30) has been characterized in response to varieties of stimuli as well as in knock out model. Our earlier data suggest that thyroid hormone 3, 3'5 Triiodo L Thyronine (T(3)), represses SMP30 in rat liver.. In highly metastatic MCF-7, human breast cancer cell line T3 treatment repressed SMP30 expression leading to enhanced apoptosis. Analysis by flow cytometry and other techniques revealed that overexpression and silencing of SMP30 in MCF-7 resulted in decelerated and accelerated apoptosis respectively. In order to identify the cis-acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected hSMP30 promoter deletion reporter vectors in MCF-7 cells. As opposed to the expected epigenetic outcome, thyroid hormone down regulated hSMP30 promoter activity despite enhanced recruitment of acetylated H3 on thyroid response elements (TREs). From the stand point of established epigenetic concept we have categorised these two TREs as negative response elements. Our attempt of siRNA mediated silencing of TRβ, reduced the fold of repression of SMP30 gene expression. In presence of thyroid hormone, Trichostatin- A (TSA), which is a Histone deacetylase (HDAC) inhibitor further inhibited SMP30 promoter activity. The above findings are in support of categorisation of both the thyroid response element as negative response elements as usually TSA should have reversed the repressions.. This is the first report of novel mechanistic insights into the remarkable downregulation of SMP30 gene expression by thyroid hormone which in turn induces apoptosis in MCF-7 human breast cancer cells. We believe that our study represents a good ground for future effort to develop new therapeutic approaches to challenge the progression of breast cancer.

Topics: Apoptosis; Binding Sites; Breast Neoplasms; Calcium-Binding Proteins; Down-Regulation; Gene Expression Regulation, Neoplastic; HEK293 Cells; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Neoplasm Metastasis; Promoter Regions, Genetic; Response Elements; Selective Estrogen Receptor Modulators; Thyroid Gland; Thyroid Hormone Receptors beta; Transcription, Genetic; Transcriptional Activation; Triiodothyronine, Reverse

CBP/p300-interacting transactivator with ED-rich carboxy-terminal domain 4 (CITED4) inhibits HIF-1α transactivation by binding to CBP/p300. We hypothesised that either somatic mutation or hypermethylation of the CITED4 gene underlies CITED4 down-regulation and thus enhanced HIF-1α expression in some breast tumours. DNA sequencing was used to screen for somatic mutations. Methylation-sensitive high resolution melting was performed to identify CITED4 methylation. RT-qPCR was carried out to measure the expression of CITED4 and selected HIF downstream targets. HIF-1α and downstream gene expression was assessed with immunohistochemistry. No somatic mutations of CITED4 were identified in 10 tumour cell lines and 100 breast carcinomas. However, CITED4 promoter methylation was identified in 5/168 breast carcinomas (four infiltrating ductal carcinomas and one infiltrating lobular carcinoma) and in 3/10 breast cancer cell lines (MDA-MB-453, MDA-MB-231 and Hs578T). CITED4 mRNA expression in cell lines was inversely correlated with DNA methylation. CITED4 mRNA expression was significantly increased in all three cell lines after 5-aza-2-deoxycytidine (DAC) treatment. Treatment of the MDA-MB-231 cell line with DAC followed by hypoxia (0.1% O²) resulted in down-regulation of expression of the HIF-1α downstream genes VEGFA and SLC2A1 (P = 0.0029). HIF-1α downstream SLC2A1 was decreased (P = 0.021) after CITED4 was re-expressed under hypoxia. Loss of expression of CITED4 in breast cancer may be due to DNA methylation but is unlikely to be due to mutation. Demethylation and histone modification can potentially reactivate CITED4 gene expression in some breast cancers and lead to changes in tumour behaviour. Strategies such as HDAC inhibitors may overcome this effect.

Topics: Azacitidine; Breast Neoplasms; Cell Line, Tumor; Decitabine; DNA Methylation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Glucose Transporter Type 1; HCT116 Cells; HL-60 Cells; Humans; Hydroxamic Acids; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; K562 Cells; Mutation; Neoplasm Invasiveness; Polymorphism, Genetic; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors

Parathyroid hormone-related protein (PTHrP) is upregulated in primary breast cancers and a major candidate for osteoclastic bone resorption present at sites of breast cancer to bone metastases. Using a human model of mammary epithelial cell lines differing in tumorigenicity and PTHrP expression, we investigated the role of epigenetic modifications for PTHrP expression. Quantitative analysis of the DNA methylation patterns at a total of 104 CpGs in the promoter region of PTHrP by pyrosequencing showed the absence of methylation in all analyzed cell lines in the large CpG island upstream of exon 1C. In the second intron of promoter 2 (P2) a region was identified containing 4 CpG nucleotides for which differential methylation correlated with the PTHrP expression level. The functional importance of this control mechanism was confirmed by the ability of the demethylating agent 5'-azacytidine to induce PTHrP mRNA and iPTHrP protein expression in previously non-expressing cell lines and increase their production by metastatic NS2T2A1 cells. In particular, transcription from P2 was activated non-tumoral S1T3 cells upon treatment with 5'-azacytidine. Our findings support the hypothesis that the methylation status of specific CpG dinucleotides is the dominant mechanism involved in silencing of PTHrP expression rather than the overall methylation of the CpG island. Methylation of the PTHrP P2 is a potential marker of breast cancer progression and might be used to evaluate the metastatic potential of breast tumors.

Topics: 5' Flanking Region; Azacitidine; Breast Neoplasms; Cells, Cultured; CpG Islands; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Neoplasm Invasiveness; Parathyroid Hormone-Related Protein; Promoter Regions, Genetic

Epigenetic mechanisms are frequently deregulated in cancer cells and can lead to the silencing of genes with tumor suppressor activities. The isoform A of the Ras-association domain family member 1 (RASSF1A) gene is one of the most frequently silenced transcripts in human tumors, however, few studies have simultaneously investigated epigenetic abnormalities associated with the 3p21.3 tumor suppressor gene cluster flanking RASSF1 (i.e., SEMA3B, HYAL3, HYAL2, HYAL1, TUSC2, RASSF1, ZMYND10, NPRL2, TMEM115, and CACNA2D2). This study aimed to investigate the role of epigenetic changes to these genes in seventeen breast cancer cell lines and in three non-tumorigenic epithelial breast cell lines (184A1, 184B5, and MCF 10A) and to evaluate the effect on gene expression of treatment with the demethylating agent 5-Aza-2'-deoxycytidine and/or Trichostatin A (TSA), a histone deacetylase inhibitor. We report that, although the RASSF1A isoform was determined to be epigenetically silenced in 15 of the 17 breast cancer cell lines, all the cell lines expressed the RASSF1C isoform. Five breast cancer cell lines overexpressed RASSF1C, when compared to the normal epithelial cell line 184A1. Furthermore, the genes HYAL1 and CACNA2D2 were significantly overexpressed after the treatments. After the combinated treatment, RASSF1A re-expression was accompanied by an increase in expression levels of the flanking genes. The Spearman's correlation coefficient indicated a positive co-regulation of the following gene pairs: RASSF1 and TUSC2 (r=0.64, p=0.002), RASSF1 and ZMYND10 (r=0.58, p=0.07), RASSF1 and NPRL2 (r=0.48, p=0.03), ZMYND10 and NPRL2 (r=0.71; p=0,0004), and NPRL2 and TMEM115 (r=0.66, p=0.001). Interestingly, the genes TUSC2, NPRL2 and TMEM115 were found to be unmethylated in each of the untreated cell lines. Chromatin immunoprecipitation using antibodies against the acetylated and trimethylated lysine 9 of histone H3 demonstrated low levels of histone methylation in these genes, which are located closest to RASSF1. These results provide evidence that epigenetic repression is involved in the down-regulation of multiple genes at 3p21.3 in breast cancer cells.

Topics: Acetylation; Azacitidine; Breast Neoplasms; Cell Line; Cell Line, Tumor; Chromosomes, Human, Pair 3; Decitabine; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histones; Humans; Hydroxamic Acids; Lysine; Multigene Family; Promoter Regions, Genetic; Tumor Suppressor Proteins

To understand the mechanism of transcriptional down-regulation of BRCA1 by promoter methylation, we screened 51 breast cancer cell lines and identified HCC38 as another BRCA1 promoter-methylated cell line in addition to UACC3199. There was low expression of BRCA1 mRNA and BRCA1 protein in both cell lines as measured by quantitative RT-PCR and western blot analysis. After transient treatment with 5-aza-2'-deoxycytidine (5-aza-CdR) and trichostatin A (TSA), re-expression of BRCA1 mRNA and BRCA1 protein was detected in UACC3199 cells, but not in HCC38 cells. Another demethylating agent, zebularine, did not induce BRCA1 re-expression in either cell line. To test the hypothesis that methylation of CpG sites may affect accessibility of the BRCA1 promoter to transcription factors and consequently cause down-regulation of BRCA1, we analyzed the binding of four transcription factors (CTCF, Sp1, E2F1 and E2F6) to the BRCA1 promoter using chromatin immunoprecipitation assay (ChIP) and quantitative PCR. CTCF and E2F1 were enriched at the unmethylated BRCA1 promoter in MCF-7 cells. In contrast, these two transcription factors were not enriched at the methylated BRCA1 promoter in UACC3199 and HCC38 cells. Following demethylating drug treatment, E2F1 was enriched at the BRCA1 promoter in the demethylated UACC3199 cells. This indicates that reduced accessibility of transcription factors to the methylated promoter is one of the mechanisms for down-regulation of BRCA1 in heavily methylated cancer cells.

Topics: Adenocarcinoma; Azacitidine; BRCA1 Protein; Breast Neoplasms; Cell Line, Tumor; Chromatin Immunoprecipitation; CpG Islands; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Genes, BRCA1; Humans; Hydroxamic Acids; Neoplasm Proteins; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Transcription Factors; Transcription, Genetic

We previously demonstrated that the PPARgamma agonist Troglitazone (TRG), a potent antiproliferative agent, in combination with the anthracycline antibiotic Doxorubicin (DOX), is an effective killer of multiple drug resistant (MDR) human cancer cells. Cell killing was accompanied by increased global histone H3 acetylation. Presently, we investigated the epigenetic and cell killing effects of TRG in estrogen receptor (ER) positive MCF7 breast cancer cells. MCF7 cells were treated with the Thiazolidinediones (TZDs) TRG and Ciglitazone (CIG), the non-TZD PPARgamma agonist 15PGJ2, and the histone deacetylase inhibitors (HDACi's) Trichostatin A (TSA), sodium butyrate and PXD101. Using MTT cell viability assays, Western analyzes and mass spectrometry, we showed a dose-dependent increase in cell killing in TRG and HDACi treated cells, that was associated with increased H3 lysine 9 (H3K9) and H3K23 acetylation, H2AX and H3S10 phosphorylation, and H3K79 mono- and di-methylation. These effects were mediated through an ER independent pathway. Using HDAC activity assays, TRG inhibited HDAC activity in cells and in cell lysates, similar to that observed with TSA. Furthermore, TRG and TSA induced a slower migrating HDAC1 species that was refractory to HDAC2 associations. Lastly, TRG and the HDACi's decreased total and phosphorylated AKT levels. These findings suggest that TRG's mode of killing may involve downregulation of PI3K signaling through HDAC inhibition, leading to increased global histone post-translational modifications.

Topics: Acetylation; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Butyrates; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromans; Dose-Response Relationship, Drug; Epigenesis, Genetic; Female; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Methylation; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Receptors, Estrogen; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfonamides; Thiazolidinediones; Troglitazone

Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1alpha dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.

Topics: Base Sequence; Breast Neoplasms; Cell Cycle Proteins; Cell Hypoxia; Cell Proliferation; Disulfides; E1A-Associated p300 Protein; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Histone Deacetylases; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Indole Alkaloids; Intercellular Signaling Peptides and Proteins; Luciferases; Molecular Sequence Data; Ovarian Neoplasms; Response Elements; Time Factors

Agents capable of increasing radioiodine concentration by stimulating the sodium/iodide symporter (NIS) expression have been extensively investigated for the treatment of certain well-differentiated breast cancers.. In this study, we analyzed the regulation of the NIS and lactoperoxidase (LPO) gene expression in 4 different human breast cancer cell lines, representative of different histotypes of breast cancer.. MCF-7, T-47D, MDA-MB231, and HCC-1937 (the latter carrying the BRCA-1 mutation) were exposed to different stimulators and the levels of NIS and LPO mRNA measured by a quantitative RT-PCR.. All-trans-Retinoic Acid (RA), Dexamethasone (DEX), Trichostatin A (TSA), and Sodium Butyrate (NaB) induced the expression of NIS mRNA in MCF-7 and T-47D cell lines, whereas HCC-1937 and MBA-MB231 were slightly responsive only to the histone-deacetylase inhibitors TSA and NaB. Minor stimulatory effects were detected on LPO mRNA in MCF-7 and T-47D treated with TSA and NaB or RA only in MCF-7, while no effect was detectable in the other two cell lines.. These data indicate that retinoic acid, alone or in combination with DEX, as well as HDAC-inhibitors are very promising agents for a radioiodine- based therapy in a large spectrum of breast cancers, including neoplasms from both basal and ductal cells, especially for the well-differentiated estrogen-dependent tumors. Other molecules or other drug combinations should be tested to extend the same strategy to the less differentiated and more aggressive tumor cells, including those carrying the BRCA mutation.

Topics: Breast Neoplasms; Butyrates; Cell Line, Tumor; Dexamethasone; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lactoperoxidase; RNA, Messenger; Symporters; Tretinoin

Epigenetic changes associated with promoter DNA methylation results in silencing of several tumor suppressor genes that lead to increased risk for tumor formation and for progression of the cancer.. Methylation specific PCR (MSP) and bisulfite sequencing were used for determination of proapoptotic gene Caspase 8 (CASP8) and the tumor suppressor gene maspin promoter methylation in four breast cancer and two non-tumorigenic breast cell lines. Involvement of histone H3 methylation in those cell lines were examined by CHIP assay.. The CpG sites in the promoter region of CASP8 and maspin were methylated in all four breast cancer cell lines but not in two non-tumorigenic breast cell lines. Demethylation agent 5-aza-2'-deoxycytidine (5-aza-dc) selectively inhibits DNA methyltransferases, DNMT3a and DNMT3b, and restored CASP8 and maspin gene expression in breast cancer cells. 5-aza-dc also reduced histone H3k9me2 occupancy on CASP8 promoter in SKBR3cells, but not in MCF-7 cells. Combination of histone deacetylase inhibitor Trichostatin A (TSA) and 5-aza-dc significant decrease in nuclear expression of Di-methyl histone H3-Lys27 and slight increase in acetyl histone H3-Lys9 in MCF-7 cells. CASP8 mRNA and protein level in MCF-7 cells were increased by the 5-aza-dc in combination with TSA. Data from our study also demonstrated that treatment with 5-FU caused a significant increase in unmethylated CASP8 and in CASP8 mRNA in all 3 cancer lines.. CASP8 and maspin expression were reduced in breast cancer cells due to promoter methylation. Selective application of demethylating agents could offer novel therapeutic opportunities in breast cancer.

Topics: Breast Neoplasms; Caspase 8; Cell Line, Tumor; Chromatin Immunoprecipitation; CpG Islands; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Models, Biological; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Serpins

Histone deacetylase (HDAC) inhibitors (HDACi) of the class I trichostatin A (TSA), CG1521 (CG), and PXD101 (PXD) were incorporated at a high rate (approximately 1mM) in liposomes made of egg phosphatidylcholine/cholesterol/distearoylphosphoethanolamine-polyethylenglycol(2000) (64:30:6). Physicochemical parameters (size, zeta potential, loading, stability, release kinetics) of these HDACi-loaded pegylated liposomes were optimized and their cytotoxicity (MTT test) was measured in MCF-7, T47-D, MDA-MB-231 and SkBr3 breast cancer cell lines. In MCF-7 cells, TSA and PXD were efficient inducers of proteasome-mediated estradiol receptor alpha degradation and they both affected estradiol-induced transcription (TSA>PXD) contrary to CG. Moreover, TSA most efficiently altered breast cancer cell viability as compared to the free drug, CG-liposomes being the weakest, while unloaded liposomes had nearly no cytotoxicity. Pegylated liposomes loaded with TSA or PXD remained stable in size, charge and biological activity for one month when stored at 4 degrees C. All HDACi-loaded liposomes released slowly the encapsulated drug in vitro, CG-loaded liposomes showed the slowest release kinetic. These formulations could improve the efficacy of HDACi not only in breast cancers but also in other solid tumors because most of these drugs are poor water soluble and unstable in vivo, and their administration remains a challenge.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Chemical Phenomena; Drug Carriers; Drug Delivery Systems; Drug Stability; Estradiol; Estrogen Receptor alpha; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Liposomes; Particle Size; Sulfonamides

Protein phosphorylation is one of the essential steps in cell signaling, and aberrant phosphorylation is a common event in human cancer. The expression of receptor type protein tyrosine phosphatase gamma (PTPRG) in normal breast is found to be approximately 50-60% higher than that of breast tumor tissue. Overexpression of PTPRG inhibits anchorage-independent growth and proliferation of breast cancer cells. To understand the tumor suppression characteristics of PTPRG, we studied its tumor suppressive function in an athymic mouse model and evaluated factors that can potentially regulate its expression in breast cancer cells.. To investigate the function of PTPRG in vivo, athymic nude mice were implanted with MCF-7 cells overexpressing PTPRG. For in vitro study, protein levels of cell cycle regulators, cell cycle re-entry, and the phosphorylation levels of extracellular signal-regulated protein kinases 1/2 (ERK1/2) were examined. In addition, methylation assays were conducted to investigate the epigenetic modification on the promoter of PTPRG.. Athymic nude mice bearing MCF-7 cells overexpressing PTPRG showed a reduction in tumor burden in comparison to animals implanted with MCF-7 cells transfected with vector alone. When these two cell lines were studied in an in vitro system, elevated mRNA and protein levels of cell cycle regulators, p21(cip) and p27(kip) were detected in MCF-7 cells overexpressing PTPRG compared to cells transfected with vector alone. Similarly, overexpression of PTPRG also delayed the re-entry of breast cancer cells into the cell cycle after serum starvation, and reduced the phosphorylation levels ERK1/2 in MCF-7 cells. In addition, methylation assays in PTPRG promoter in breast cancer cell lines (including SK-Br-3) revealed an aberrant methylation pattern. When SK-Br-3 and MCF-7 cells were treated with deoxy-5-azacytidine (DAC) and trichostatin A (TSA), these compounds reactivated the expression of PTPRG, suggesting an epigenetic control on its expression.. Our results indicated that PTPRG inhibited breast tumor formation in vivo; PTPRG may up-regulate p21(cip) and p27(kip) proteins through the ERK1/2 pathway. This study also showed methylation-mediated silencing of PTPRG in breast cancer cell lines. These data indicate that PTPRG exhibits the characteristics of a breast tumor suppressor.

Topics: Animals; Azacitidine; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; CpG Islands; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Decitabine; DNA Methylation; Extracellular Signal-Regulated MAP Kinases; Female; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Mice; Phosphorylation; Receptor-Like Protein Tyrosine Phosphatases, Class 5

The status of estrogen receptor-α (ERα) is critical to the clinical prognosis and therapeutic approach in breast cancer. ERα-negative breast cancer is clinically aggressive and has a poor prognosis because of the lack of hormone target-directed therapies. Previous studies have shown that epigenetic regulation plays a major role in ERα silencing in human breast cancer cells. Dietary green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), is believed to be an anticancer agent in part through its regulation of epigenetic processes.. In our current studies, we found that EGCG can reactivate ERα expression in ERα-negative MDA-MB-231 breast cancer cells. Combination studies using EGCG with the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), revealed a synergistic effect of reactivation of ERα expression in ERα-negative breast cancer cells. Reactivation of ERα expression by EGCG and TSA treatment was found to sensitize ERα-dependent cellular responses to activator 17β-estradiol (E2) and antagonist tamoxifen in ERα-negative breast cancer cells. We also found that EGCG can lead to remodeling of the chromatin structure of the ERα promoter by altering histone acetylation and methylation status thereby resulting in ERα reactivation. A decreased binding of the transcription repressor complex, Rb/p130-E2F4/5-HDAC1-SUV39H1-DNMT1, in the regulatory region of the ERα promoter also contributes to ERα transcriptional activation through treatment with EGCG and/or TSA.. Collectively, these studies show that green tea EGCG can restore ERα expression by regulating epigenetic mechanisms, and this effect is enhanced when combined with an HDAC inhibitor. This study will facilitate more effective uses of combination approaches in breast cancer therapy and will help to explore more effective chemotherapeutic strategies toward hormone-resistant breast cancer.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Catechin; Cell Line, Tumor; Cell Survival; Chromatin Immunoprecipitation; DNA Methylation; Drug Synergism; Estrogen Receptor alpha; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Polymerase Chain Reaction; Promoter Regions, Genetic; Tamoxifen

Epidemiological and experimental evidence implicates estrogens in the etiology and progression of breast cancer. The biosynthesis of estrogens from androgens is catalyzed by an enzymatic complex designated as aromatase (CYP19). Using quantitative real-time PCR and Western blot analysis, we demonstrated that trichostatin A (TSA) histone deacetylase inhibitor significantly reduced CYP19 transcript and protein contents in MCF-7 breast cancer cells. We also found that TSA lowered CYP19 transcript stability and significantly decreased the transcript's half-life from approximately 6h to 3.5h. Our results from experiments with a protein biosynthesis inhibitor suggest the involvement of an RNase and/or mRNA stabilization protein in CYP19 transcript stabilization. Since malignant tissue aromatase is a significant estrogen producer involved in breast tumor progression, our findings may have clinical implication.

Topics: Adenocarcinoma; Antineoplastic Agents, Hormonal; Aromatase; Aromatase Inhibitors; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Half-Life; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasm Proteins; RNA Stability; RNA, Messenger; RNA, Neoplasm

This study analyzes the relationship between coxsackie-adenovirus receptor (CAR) expression (transmembrane and soluble isoforms) and hormone sensitivity in 95 breast cancers. Furthermore, prognostic significance of the expression of the various CAR isoforms was investigated. In addition, inducibility of CAR expression by estradiol and tamoxifen was assessed in various breast cancer cell lines. Expression of transmembrane CAR (hCAR) highly correlated with estrogen receptivity, but was independent of the expression of progesterone receptor (PR). Furthermore, hCAR expression was significantly higher in tumors with low-grade malignancy. However, no relationship between hCAR expression and tumor size, lymph node status, or survival was revealed. In the hormone receptor-positive breast cancer cell line T47-D expression of hCAR and its soluble isoforms was increased by treatment with estradiol and tamoxifen. In contrast, no induction of either CAR isoform was achieved in receptor-negative cell lines. Furthermore, enhancement of hCAR expression was significantly greater when cells were treated with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) than when treated with estradiol or tamoxifen. Moreover, sensitivity to TSA induction of hCAR was considerably greater in receptor-positive than in receptor-negative cell lines. No additive effect on CAR expression was found when TSA was combined with either estradiol or tamoxifen. In conclusion, the so far undescribed association between estrogen receptivity and the expression of hCAR in breast cancer seems to not only reflect a phenotype of low malignancy, but expression of hCAR may also be directly influenced by ER-specific ligands.

Topics: Aged; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Constitutive Androstane Receptor; Enzyme Inhibitors; Estradiol; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Middle Aged; Protein Isoforms; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tamoxifen

Gene silencing by aberrant epigenetic chromatin alteration is a well-recognized event contributing to tumorigenesis. Although genetically engineered tumor-prone mouse models have proven a powerful tool in understanding many aspects of carcinogenesis, to date few studies have focused on epigenetic alterations in mouse tumors. To uncover epigenetically silenced tumor suppressor genes (TSGs) in mouse mammary tumor cells, we conducted initial genome-wide screening by combining the treatment of cultured cells with the DNA demethylating drug 5-aza-2'-deoxycytidine (5-azadC) and the histone deacetylase inhibitor trichostatin A (TSA) with expression microarray. By conducting this initial screen on EMT6 cells and applying protein function and genomic structure criteria to genes identified as upregulated in response to 5-azadC/TSA, we were able to identify two characterized breast cancer TSGs (Timp3 and Rprm) and four putative TSGs (Atp1B2, Dusp2, FoxJ1 and Smpd3) silenced in this line. By testing a panel of 10 mouse mammary tumor lines, we determined that each of these genes is commonly hypermethylated, albeit with varying frequency. Furthermore, by examining a panel of human breast tumor lines and primary tumors we observed that the human orthologs of ATP1B2, FOXJ1 and SMPD3 are aberrantly hypermethylated in the human disease whereas DUSP2 was not hypermethylated in primary breast tumors. Finally, we examined hypermethylation of several genes targeted for epigenetic silencing in human breast tumors in our panel of 10 mouse mammary tumor lines. We observed that the orthologs of Cdh1, RarB, Gstp1, RassF1 genes were hypermethylated, whereas neither Dapk1 nor Wif1 were aberrantly methylated in this panel of mouse tumor lines. From this study, we conclude that there is significant, but not absolute, overlap in the epigenome of human and mouse mammary tumors.

Topics: Animals; Azacitidine; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Databases, Genetic; Decitabine; DNA Methylation; DNA Modification Methylases; Epigenesis, Genetic; Gene Expression Regulation; Gene Silencing; Genes, Tumor Suppressor; Glycoproteins; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mammary Neoplasms, Animal; Mice; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Structure-Activity Relationship; Tissue Inhibitor of Metalloproteinase-3

Histone deacetylation occurs upon the transfer of somatic nuclei into enucleated oocytes, but its role in reprogramming somatic chromatin to the totipotent state is unknown. To investigate the importance of histone deacetylation in reprogramming, we constructed embryos by electrofusing breast cancer cells with enucleated mouse oocytes. The reconstructed embryos were then cultured before and/or after activation for 6h in the presence of trychostatin A (TSA), a potent inhibitor of histone deacetylase. Total RNA was isolated from these TSA-treated and untreated embryos and real-time reverse transcription PCR was conducted to monitor transcription of ErbB2, Muc1, eIF-4C, MuERV-L, and c-mos genes. The nuclear-cytoplasmic interaction inhibited typical expression of ErbB2 and Muc1 in the somatic cells. Moreover, the inhibition of histone deacetylation prior to activation did not increase the levels of eIF-4C, MuERV-L, and c-mos expression in the nuclear transfer (NT) embryos (P>0.05), whereas additional treatment with 100nM TSA beyond the activation point improved expression of these genes (P<0.05). Trychostatin A treatment also improved the development rates of NT embryos at the 2-cell, 4-cell, and blastocyst stages (78.6% vs. 90.2%, 45.2% vs. 68.9%, and 16.7% vs. 30.3%, respectively, P<0.05). We hypothesized that the reprogramming of gene expression in NT embryos is independent of somatic histone deacetylation, and that hyperacetylation may have a positive effect on NT embryo development.

Topics: Animals; Breast Neoplasms; Cell Nucleus; Cells, Cultured; Cellular Reprogramming; Cloning, Organism; Embryo Culture Techniques; Embryo, Mammalian; Embryonic Development; Female; Fertilization in Vitro; Gene Expression Regulation, Developmental; Hydroxamic Acids; Male; Mice; Mice, Inbred ICR; Nuclear Transfer Techniques; Pregnancy

A growing number of studies indicate the importance of the lysyl oxidase family in the promotion of epithelial neoplasms towards their more aggressive forms. However, the role of individual family members in carcinoma progression has yet to be ascertained. In this study, we analyzed LOXL2 expression in malignantly transformed MCF-7 and normal MCF-10A mammary epithelial cell line clones stably transduced with LOXL2 in vitro, and in normal and cancerous breast tissue samples in vivo. We found LOXL2 to be catalytically active in both MCF-7 and MCF-10 clones. LOXL2 overexpression promoted a more mesenchymal morphology in both cell types, but LOXL2-induced increase in migratory ability could only be established in MCF-7 clones. We demonstrated altered localization of the LOXL2 protein in breast cancer tissue compared to normal mammary tissue, and altered localization and processing of LOXL2 protein in breast cancer cell lines compared to normal cell lines, which may allow LOXL2 to interact with different intra and extracellular components during tumor progression. Results support the role of LOXL2 in selectively promoting a metastatic phenotype in breast tumor cells. Additional data suggest epigenetic molecular mechanisms in tumor specific regulation of LOXL2 expression that could be explored as a molecular target in the prevention of breast cancer progression.

Topics: Amino Acid Oxidoreductases; Azacitidine; Blotting, Western; Breast Neoplasms; Catalysis; Cell Line; Cell Line, Tumor; Decitabine; Gene Expression Regulation, Enzymologic; Humans; Hydroxamic Acids; Immunohistochemistry; Mammary Glands, Human; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction

CDKN1C (encoding tumor suppressor p57(KIP2)) is a cyclin-dependent kinase (CDK) inhibitor whose family members are often transcriptionally downregulated in human cancer via promoter DNA methylation. In this study, we show that CDKN1C is repressed in breast cancer cells mainly through histone modifications. In particular, we show that CDKN1C is targeted by histone methyltransferase EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3), and can be strongly activated by inhibition of EZH2 in synergy with histone deacetylase inhibitor. Consistent with the overexpression of EZH2 in a variety of human cancers including breast cancer, CDKN1C in these cancers is downregulated, and breast tumors expressing low levels of CDKN1C are associated with a poor prognosis. We further show that assessing both EZH2 and CDKN1C expression levels as a measurement of EZH2 pathway activity provides a more predictive power of disease outcome than that achieved with EZH2 or CDKN1C alone. Taken together, our study reveals a novel epigenetic mechanism governing CDKN1C repression in breast cancer. Importantly, as a newly identified EZH2 target with prognostic value, it has implications in patient stratification for cancer therapeutic targeting EZH2-mediated gene repression.

Topics: Breast Neoplasms; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p57; DNA Methylation; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Epigenesis, Genetic; Humans; Hydroxamic Acids; Polycomb Repressive Complex 2; Polymerase Chain Reaction; Promoter Regions, Genetic; Transcription Factors

Transcriptional activity of NF-kappaB is inhibited by the liganded glucocorticoid receptor (GR), which exists mainly in two splice variants as functional GRalpha and nonfunctional GRbeta. We investigated the effect of 5-aza-2'-deoxycytidine (5-dAzaC), trichostatin A (TSA), and sodium butyrate (NaBu) on GRalpha,GRbeta and ASF/SF2 splicing factor expression in HT-29 colon and MCF-7 breast carcinoma cells.. HT-29 and MCF-7 cells were cultured in the absence or in the presence of 5-dAzaC, TSA, and NaBu, followed by RNA and protein isolation. The transcript and protein levels of GRalpha, GRbeta ASF/SF2 were determined by reverse transcription, real-time quantitative PCR and Western blot analysis.. We found that 5-dAzaC, TSA, and NaBu lead to an increase in GRalpha and ASF/SF2 transcript levels and a decrease in GRbeta transcript levels in HT-29 and MCF-7 cells. The 5-dAzaC, TSA, and NaBu resulted in increased GRalpha and ASF/SF2 protein levels and GRbeta protein downregulation in HT-29 cells. The most increased GRalpha protein expression in MCF-7 cells was observed with NaBu. However, all of these compounds inhibited GRbeta protein expression in MCF-7 cells. The MCF-7 cells treated with NaBu demonstrated a remarkable increase in ASF/SF2 protein expression.. Because NF-kappaB is considered to be a factor in the augmentation of malignant properties of cells, treatment of tumors with 5-dAzaC, TSA, and NaBu may provide a novel approach to the enhancement of therapeutic effects of glucocorticoids in epithelial carcinomas.

Topics: Azacitidine; Breast Neoplasms; Butyrates; Colonic Neoplasms; Decitabine; Enzyme Inhibitors; HT29 Cells; Humans; Hydroxamic Acids; Nuclear Proteins; Receptors, Glucocorticoid; RNA-Binding Proteins; Serine-Arginine Splicing Factors

Transcriptional regulation of estrogen receptor-alpha (ERalpha) involves both epigenetic mechanisms and trans-active factors, such as TFAP2C, which induces ERalpha transcription through an AP-2 regulatory region in the ERalpha promoter. Attempts to induce endogenous ERalpha expression in ERalpha-negative breast carcinomas by forced overexpression of TFAP2C have not been successful. We hypothesize that epigenetic chromatin structure alters the activity of TFAP2C at the ERalpha promoter.. DNA methylation, histone acetylation, and chromatin accessibility were examined at the ERalpha promoter in a panel of breast carcinoma cell lines. TFAP2C and polymerase II binding were analyzed by chromatin immunoprecipitation. Epigenetic chromatin structure was altered using drug treatment with 5-aza-2'-deoxycytidine (AZA) and trichostatin A (TSA).. The ERalpha promoter in the ERalpha-negative lines MDA-MB-231, MCF10A, and MCF7-5C show CpG island methylation, histone 3 lysine 9 deacetylation, and decreased chromatin accessibility compared with ERalpha-positive cell lines MCF7 and T47-D. Treatment with AZA/TSA increased chromatin accessibility at the ERalpha promoter and allowed TFAP2C to induce ERalpha expression in ERalpha-negative cells. Chromatin immunoprecipitation analysis showed that binding of TFAP2C to the ERalpha promoter is blocked in ERalpha-negative cells but that treatment with AZA/TSA enabled TFAP2C and polymerase II binding.. We conclude that the activity of TFAP2C at specific target genes depends upon epigenetic chromatin structure. Furthermore, the combination of increasing chromatin accessibility and inducing TFAP2C provides a more robust activation of the ERalpha gene in ERalpha-negative breast cancer cells.

Topics: Acetylation; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Chromatin; Chromatin Immunoprecipitation; CpG Islands; Decitabine; DNA Methylation; Enzyme Inhibitors; Estrogen Receptor alpha; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Lysine; Promoter Regions, Genetic; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor AP-2

Although the p53 anti-oncogene is an important target for gene therapy of cancer, some cancers are resistant to p53 gene transfer. For this reason, it is important to find effective drugs to enhance cytotoxic effects of p53 gene transfer. Recent reports demonstrated that some histone deacetylase inhibitors in combination with p53 gene therapy induced apoptosis in certain cancer cells more efficiently than p53 gene therapy alone. We investigated whether histone deacetylase inhibitor trichostatin A (TSA), in combination with p53 gene transfer could synergistically induce apoptosis in the breast cancer cell line MDA-MB-231. Whereas the adenovirus-expressing p53 (Ad-p53) by itself at up to 100 multiplicity of infection (MOI) induced apoptosis at a low level, Ad-p53 in combination with TSA synergistically induced apoptosis at a higher level in MDA-MB-231 cells than TSA or Ad-p53 alone. However, the combination of Ad-p53 and TSA did not enhance the expressions of p53 or p53-induced genes that are involved in apoptosis, and synergistically reduced the mitochondrial membrane potential and enhanced caspase-3 activity. These results suggest that TSA have synergistic effects on the induction of apoptosis in MDA-MB-231 cells when combined with p53 gene transfer.

Topics: Adenoviridae; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Caspase 3; Cell Line, Tumor; Chemotherapy, Adjuvant; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Genetic Therapy; Genetic Vectors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Membrane Potential, Mitochondrial; RNA, Messenger; Transfection; Tumor Suppressor Protein p53

The antimalarial drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have potential applications in cancer treatment. The growth of MCF-7 and MDA-MB-231 human breast cancer cells in vitro was inhibited by CQ and HCQ and these cells were more sensitive than nontumorigenic MCF-10A breast epithelial cells. Furthermore, all-trans retinoic acid (ATRA) augmented the anticancer effects of CQ and HCQ as evidenced by significant reductions in Ki67-positive cancer cells and clonogenicity compared with cells treated with CQ or HCQ in the absence of ATRA. As an earlier study suggested that CQ, HCQ, and ATRA are breast cancer cell differentiation agents, these agents were screened in cell-free histone deacetylase (HDAC) and histone acetyltransferase (HAT) assays. ATRA, but not CQ or HCQ, inhibited HDAC activity in HeLa nuclear extracts. Growth inhibitory concentrations of HCQ and ATRA stimulated purified p300/CBP-associated factor, where CBP is the cAMP-response element binding protein, HAT activity. To investigate whether growth inhibitory concentrations of these agents influenced protein acetylation in cells, gel-purified histone H3 and histone H4 were analyzed using mass spectrometry. HCQ alone and HCQ+ATRA treatments altered the acetylation status in the N-terminal lysines of histones H3 and H4 compared with dimethyl sulfoxide (DMSO) controls. The results indicated that HCQ and ATRA regulate protein acetylation events in MCF-7 breast cancer cells, and identify a potential mechanism for their effects on breast cancer cell growth and differentiation.

Topics: Acetylation; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Cellular Senescence; Chloroquine; Enzyme Inhibitors; Female; HeLa Cells; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Hydroxychloroquine; Ki-67 Antigen; Mass Spectrometry; Tretinoin; Tumor Stem Cell Assay

Deletion of 11q23-q24 is frequent in a diverse variety of malignancies, including breast and colorectal carcinoma, implicating the presence of a tumor suppressor gene at that chromosomal region. We examined a 6-Mb region on 11q23 by high-resolution deletion mapping, using both loss of heterozygosity analysis and customized microarray comparative genomic hybridization. LARG (leukemia-associated Rho guanine-nucleotide exchange factor) (also called ARHGEF12), identified from the analysed region, is frequently underexpressed in breast and colorectal carcinomas with a reduced expression observed in all breast cancer cell lines (n=11), in 12 of 38 (32%) primary breast cancers, 5 of 10 (50%) colorectal cell lines and in 20 of 37 (54%) primary colorectal cancers. Underexpression of the LARG transcript was significantly associated with genomic loss (P=0.00334). Hypermethylation of the LARG promoter was not detected in either breast or colorectal cancer, and treatment of four breast and four colorectal cancer cell lines with 5-aza-2'-deoxycytidine and/or trichostatin A did not result in a reactivation of LARG. Enforced expression of LARG in breast and colorectal cancer cells by stable transfection resulted in reduced cell proliferation and colony formation, as well as in a markedly slower cell migration rate in colorectal cancer cells, providing functional evidence for LARG as a candidate tumor suppressor gene.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 11; Colorectal Neoplasms; Decitabine; DNA Methylation; Female; Guanine Nucleotide Exchange Factors; Humans; Hydroxamic Acids; Male; Nucleic Acid Hybridization; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Rho Guanine Nucleotide Exchange Factors; Transfection; Tumor Suppressor Proteins

Histone deacetylase inhibitors (HDACi) show promise as a novel class of antitumoral agents and have shown the ability to induce apoptosis of tumor cells. To gain a better understanding of the action of HDACi, we conducted a functional gene screen approach named suppression of mortality by antisense rescue technique to identify the key genes responsible for the tumor-selective killing trichostatin A. Over 20 genes associated with HDACi-induced mortality were identified. One of the confirmed positive hits is LIV1, a putative zinc transporter. LIV1 is significantly induced by treatment with HDACi in a number of tumor cells, but not in normal cells. Knockdown of LIV1 suppressed apoptosis induced by HDACi in tumor cells. Although HDACi induced a slight increase in the free intracellular zinc concentration, knockdown of LIV1 significantly enhanced the intracellular zinc level, which was associated with resistance to apoptosis. On the other hand, pretreatment of the cells with a specific zinc chelator TPEN reversed the apoptosis resistance conferred by knockdown of LIV1. However, the biological effects of TPEN were abolished by addition of physiologic concentrations of zinc. Taken together, the present study identifies LIV1 as a critical mediator responsible for HDACi-induced apoptosis. The effect of LIV1 is, at least in part, mediated by affecting intracellular zinc homeostasis, which may be related to alteration of the catalytic activity of the Caspase 3 and expression of some BCL-2 family genes. As such, these findings highlight a novel mechanism underlying the action of HDACi that could be potentially useful in the clinical setting.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cation Transport Proteins; Cell Death; Cell Growth Processes; Cell Line, Tumor; DNA, Antisense; Drug Synergism; Female; Gene Knockdown Techniques; HeLa Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Neoplasm Proteins; Proto-Oncogene Proteins c-bcl-2; Transfection; Uterine Cervical Neoplasms; Zinc

Polycomb protein EZH2-mediated gene silencing is implicated in breast tumorigenesis through methylation of histone H3 on Lysine 27 (H3K27). We have previously shown that S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A can modulate histone methylation and disrupt EZH2 complex. Here, we used 3-deazaneplanocin A, together with other chromatin remodeling agents, as well as RNA interference-mediated EZH2 depletion, to probe the role of EZH2 in coordination with other epigenetic components in gene regulation in breast cancer cells. Through genome-wide gene expression analysis, coupled with extensive chromatin immunoprecipitation analysis of histone modifications, we have identified a variety of gene sets that are regulated either by EZH2 alone or through the coordinated action of EZH2 with HDAC and/or DNA methylation. We further found that tumor antigen GAGEs were regulated by distinct epigenetic mechanisms in a cell context-dependent manner, possibly reflecting mechanistic heterogeneity in breast cancer. Intriguingly, we found that EZH2 regulates a remarkable cohort of genes whose functions are highly enriched in immunoresponse and autocrine inflammation network, and that their transcriptional activation upon EZH2 perturbation is cancer specific, revealing a potential novel role of EZH2 in regulating cancer immunity. These findings show the complexity and diversity of epigenetic regulation in human cancer and underscore the importance for developing combinatorial pharmacologic approaches for effective epigenetic gene reactivation.

Topics: Adenosine; Antigens, Neoplasm; Azacitidine; Breast Neoplasms; Cell Line; Cell Line, Tumor; Chromatin Immunoprecipitation; Cluster Analysis; CpG Islands; Decitabine; DNA Methylation; DNA-Binding Proteins; Enhancer of Zeste Homolog 2 Protein; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunoblotting; Methylation; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Polycomb Repressive Complex 2; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transcription Factors

XB-S is an amino-terminal truncated protein of tenascin-X (TNX) in humans. The levels of the XB-S transcript, but not those of TNX transcripts, were increased upon hypoxia. We identified a critical hypoxia-responsive element (HRE) localized to a GT-rich element positioned from -1410 to -1368 in the XB-S promoter. Using an electrophoretic mobility shift assay (EMSA), we found that the HRE forms a DNA-protein complex with Sp1 and that GG positioned in -1379 and -1378 is essential for the binding of the nuclear complex. Transfection experiments in SL2 cells, an Sp1-deficient model system, with an Sp1 expression vector demonstrated that the region from -1380 to -1371, an HRE, is sufficient for efficient activation of the XB-S promoter upon hypoxia. The EMSA and a chromatin immunoprecipitation (ChIP) assay showed that Sp1 together with the transcriptional repressor histone deacetylase 1 (HDAC1) binds to the HRE of the XB-S promoter under normoxia and that hypoxia causes dissociation of HDAC1 from the Sp1/HDAC1 complex. The HRE promoter activity was induced in the presence of a histone deacetylase inhibitor, trichostatin A, even under normoxia. Our results indicate that the hypoxia-induced activation of the XB-S promoter is regulated through dissociation of HDAC1 from an Sp1-binding HRE site.

Topics: Animals; Base Sequence; Binding Sites; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Drosophila; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Histone Deacetylases; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Molecular Sequence Data; Protein Binding; Response Elements; RNA, Messenger; Sp1 Transcription Factor; Tenascin; Transcriptional Activation

Many breast cancer cells typically exhibit lower expression of manganese superoxide dismutase (MnSOD) compared to the normal cells from which they arise. This decrease can often be attributed to a defect in the transcription of SOD2, the gene encoding MnSOD; however, the mechanism responsible for this change remains unclear. Here, we describe how altered histone modifications and a repressive chromatin structure constitute an epigenetic process to down regulate SOD2 in human breast carcinoma cell lines. Utilizing chromatin immunoprecipitation (ChIP) we observed decreased levels of dimethyl H3K4 and acetylated H3K9 at key regulatory elements of the SOD2 gene. Consistent with these results, we show that loss of these histone modifications creates a repressive chromatin structure at SOD2. Transcription factor ChIP experiments revealed that this repressive chromatin structure influences the binding of SP-1, AP-1, and NFkappaB to SOD2 regulatory cis-elements in vivo. Lastly, we show that treatment with the histone deacetylase inhibitors trichostatin A and sodium butyrate can reactivate SOD2 expression in breast cancer cell lines. Taken together, these results indicate that epigenetic silencing of SOD2 could be facilitated by changes in histone modifications and represent one mechanism leading to the altered expression of MnSOD observed in many breast cancers.

Topics: Acetylation; Breast Neoplasms; Cell Line, Tumor; Chromatin; Chromatin Immunoprecipitation; Female; Gene Silencing; Histone Code; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Methylation; NF-kappa B; Promoter Regions, Genetic; RNA, Messenger; Sp1 Transcription Factor; Superoxide Dismutase; Transcription Factor AP-1; Transcriptional Activation

A possible epigenetic regulation of the two isoenzymes of fructose 1,6-bisphosphatase (FBPase) was studied in liver, muscle, mamma, breast cancer and in different cancer cell lines. Results obtained after bisulfite sequencing revealed a different CpG methylation of both promoters in liver, muscle and breast tissue which is putatively involved in the cell-type specific gene expression of the two enzymes. In tumor cell lines, demethylation with 5-aza-deoxycytidine activated the expression of both isoenzymes. Additional inhibition of histone deacetylase with trichostatin A further increased FBPase mRNA concentrations. Since cancers typically have an abnormal energy metabolism and exhibit a low gluconeogenic phenotype, it was studied whether promoter methylation contributes to the decreased expression of FBPase in breast cancer. When non-malignant and malignant tissue samples from the same patient were compared a correlation between an increase of FBPase promoter methylation and a decrease of FBPase mRNA levels was observed.

Topics: Adult; Aged; Aged, 80 and over; Azacitidine; Breast Neoplasms; Cell Line, Tumor; DNA Methylation; Female; Fructose-Bisphosphatase; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hydroxamic Acids; Middle Aged; Neoplasms; Promoter Regions, Genetic

Hepatocyte growth factor (HGF), through Met receptor binding, fulfils numerous functions in invasive tumour growth (survival/proliferation, motility, apoptosis), but epigenetic control of gene expression in this process is poorly understood. In HGF-treated breast cancer cells we studied (a) the chemoinvasion towards CXCL12 (ligand of the chemokine-receptor CXCR4) and (b) the mechanistic basis, that is, the transduction pathways that regulate CXCR4-mediated invasion, and the role played by histone deacetylases (HDACs) after blockade with trichostatin A (TSA). In highly invasive and metastatic MDA-MB231 cells HGF had a dual inhibitory effect, reducing spontaneous migration and specific chemoinvasion towards CXCL12, the latter by decreasing CXCR4 transactivation and protein level. After HGF the levels of phosphorylated (therefore active) c-Src and Akt persistently increased, indicating a role of these signal transducers in the HGF-dependent cellular and molecular effects. c-Src wild-type expression vector (Srcwt) increased active c-Src and mimicked the HGF-dependent inhibition of CXCR4 transactivation. Our findings indicate that HDACs participated in the HGF-inhibitory effects. In fact, blockade of HDACs hindered the HGF- and Srcwt-dependent reductions of CXCR4 transactivation and invasiveness, while inhibition of endogenous c-Src was additive with HGF, further reducing specific chemoinvasion. In conclusion, in MDA-MB231 cells HDAC blockade with TSA partly counteracted the HGF-dependent effects through molecular events that included enhancement of the expression of the genes for invasiveness Met and CXCR4 (depending on serum conditions), reduction of endogenous phospho-c-Src/c-Src and phosphoAkt/Akt ratios and triggering of apoptosis. The potential therapeutic use of TSA should take into account the variable aggressiveness of breast carcinoma cells and microenvironment signals such as HGF at the secondary growth site of the tumour. It was interesting that HGF reduced motility and CXCR4 functionality only of MDA-MB231 cells, and not of low-invasive MCF-7 cells, suggesting a mechanism implicated in metastatic cell homing.

Topics: Breast Neoplasms; Cell Line, Tumor; Chemokine CXCL12; Enzyme Inhibitors; Hepatocyte Growth Factor; Histone Deacetylases; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Neoplasm Invasiveness; Receptors, CXCR4; Signal Transduction

This study examined the mechanism for the anti-cancer effects of histone deacetylase (HDAC) inhibitor trichostatin A (TsA) in H-ras-transformed human breast epithelial (MCF10A-ras) cells. The effects of TsA on anti-cancer effects of MCF10A-ras cells were determined by measuring the level of cell cycle regulator expression and apoptotic cell death using Western blotting and flow cytometry analysis, respectively. TsA induced morphological changes, apoptotic cell death and modulation of the cell cycle regulatory proteins in the MCF10A-ras cells. TsA increased the levels of acetylated histone H3 and H4 in MCF10A-ras cells. In addition, TsA markedly down-regulated the expression of cyclin D1 and CDK4, up-regulated the expression of p21WAF1 and p53 and induced cell cycle arrest at the G1 phase in MCF10A-ras cells. The levels of hyperphosphorylation of the Rb protein were lower in MCF10A-ras cells after the TsA treatment. Furthermore, the up-regulation of p53 promoted Bax expression, which led to the activation of pro-caspase-3 and eventually to apoptosis in MCF10A-ras cells. TsA significantly increased the levels of ERK1/2 phosphorylation in MCF10A-ras cells. Overall, the TsA-activated ERK pathway plays an important role in cell cycle arrest and apoptosis through the ERK-dependent induction of p21 in Ras-related human cancer cells.

Topics: Apoptosis; bcl-2-Associated X Protein; Breast Neoplasms; Caspase 3; Cell Cycle Proteins; Cell Line, Transformed; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; G1 Phase; Gene Expression Regulation; Genes, ras; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mammary Glands, Human; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Tumor Suppressor Protein p53

Trichostatin A (TSA) and 5-Aza 2'deoxycytidine (AZA), two well characterized pharmacologic inhibitors of histone deacetylation and DNA methylation, affect estrogen receptor alpha (ER) levels differently in ER-positive versus ER-negative breast cancer cell lines. Whereas pharmacologic inhibition of these epigenetic mechanisms results in re-expression and increased estrogen receptor alpha (ER) levels in ER-negative cells, treatment in ER-positive MCF7 cells results in decreased ER mRNA and protein levels. This decrease is dependent upon protein interaction with the ER 3'UTR. Actinomycin D studies showed a 37.5% reduction in ER mRNA stability from 4 to 1.5 h in AZA/TSA treated MCF7 cell lines; an effect not seen in 231ER + cells transfected with the ER coding region but lacking incorporation of the 3'UTR. AZA/TSA do not appear to directly interact with the 3'UTR but rather decrease stability through altered subcellular localization of the RNA binding protein, HuR. siRNA inhibition of HuR expression reduces both the steady-state and stability of ER mRNA, suggesting that HuR plays a critical role in the control of ER mRNA stability. Our data suggest that epigenetic modulators can alter stability through modulation of HuR subcellular distribution. Taken together, these data provide a novel anti-estrogenic mechanism for AZA and TSA in ER positive human breast cancer cells.

Topics: 3' Untranslated Regions; Antigens, Surface; Azacitidine; Base Sequence; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Decitabine; ELAV Proteins; ELAV-Like Protein 1; Enzyme Inhibitors; Epigenesis, Genetic; Estrogen Receptor alpha; Estrogen Receptor Modulators; Female; Humans; Hydroxamic Acids; Immunoprecipitation; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA Stability; RNA-Binding Proteins; RNA, Messenger; Transfection

The anti-proliferative effects of histone deacetylase (HDAC) inhibitors and 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] converge via the interaction of un-liganded vitamin D receptor (VDR) with co-repressors recruiting multiprotein complexes containing HDACs and via the induction of cyclin-dependent kinase inhibitor (CDKI) genes of the INK4 and Cip/Kip family. We investigated the effects of the HDAC inhibitor Trichostatin A (TSA) and 1alpha,25(OH)2D3 on the proliferation and CDKI gene expression in malignant and non-malignant mammary epithelial cell lines. TSA induced the INK4-family genes p18 and p19, whereas the Cip/Kip family gene p21 was stimulated by 1alpha,25(OH)2D3. Chromatin immunoprecipitation and RNA inhibition assays showed that the co-repressor NCoR1 and some HDAC family members complexed un-liganded VDR and repressed the basal level of CDKI genes, but their role in regulating CDKI gene expression by TSA and 1alpha,25(OH)2D3 were contrary. HDAC3 and HDAC7 attenuated 1alpha,25(OH)2D3-dependent induction of the p21 gene, for which NCoR1 is essential. In contrast, TSA-mediated induction of the p18 gene was dependent on HDAC3 and HDAC4, but was opposed by NCoR1 and un-liganded VDR. This suggests that the attenuation of the response to TSA by NCoR1 or that to 1alpha,25(OH)2D3 by HDACs can be overcome by their combined application achieving maximal induction of anti-proliferative target genes.

Topics: Acetylation; Antineoplastic Agents; Breast; Breast Neoplasms; Calcitriol; Cell Line; Cell Line, Tumor; Chromatin; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor Proteins; Enzyme Inhibitors; Epithelial Cells; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Promoter Regions, Genetic; Receptors, Calcitriol; Repressor Proteins; RNA Interference; Transcription Initiation Site; Transcription, Genetic

Histone deacetylase inhibitors (HDACi) are a new class of anticancer agents that cause growth arrest, differentiation and/or apoptosis in many tumor cells. As acetylation regulates the activity of the anti-apoptotic transcription factor NF-kappaB, we investigated whether the proteasome inhibitor MG-132 would inhibit NF-kappaB activation and as a consequence potentiate HDACi-dependent apoptosis in breast cancer cells. We observed that the HDACi suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA) induced cell death but also enhanced NF-kappaB-activity. This increase of NF-kappaB activity was strongly reduced by the addition of MG-132. Moreover, MG-132 potentiates the HDACi-induced cell death that was associated with caspase-3 activation, and PARP cleavage. Induction of the stress related kinases JNK and p38 and the up-regulation of p21 and p27 were also observed after co-treatment of cells with HDACi and MG-132. Disruption of the NF-kappaB pathway by BAY 11-7085 or IkappaB-SR mimicked the action of MG-132 in promoting HDACi-induced cell death. Thus, the combined treatment with HDACi and proteasome inhibitors potentiates apoptosis in breast cancer cells representing a novel strategy for breast cancer therapy.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Colony-Forming Units Assay; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Female; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; I-kappa B Proteins; Leupeptins; Luciferases; Membrane Potential, Mitochondrial; NF-kappa B; NF-KappaB Inhibitor alpha; Nitriles; Sulfones; Transfection; Tumor Cells, Cultured; Vorinostat

Estrogen receptor alpha (ER alpha) mediates the growth stimulation of estrogen in breast cancer cells and is a useful predictive factor for response to endocrine therapy. It is reported that ER alpha was induced in ER alpha negative breast cancer cells by both DNA methyltransferase-1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (AZA) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, whether the breast cancer cells with induced ER alpha restore response to endocrine therapy requires to be further researched.. Reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to explore the change in the mRNA of ER alpha, PR and pS2 in the ER alpha negative breast cancer cells MDA-MB-435 treated with two chemicals (AZA + TSA). Water-soluble tetrazolium salt-8 (WST-8) method was used to study the proliferation rate of the breast cancer cells. Flow cytometer (FCW) was used to analyze the distribution of cell cycle of these breast cancer cells. Some xenograft models in nude mice were used to further study the results we found in vitro.. In this study we observed that the mRNA of ER alpha, PR and pS2 in the ER alpha negative breast cancer cells MDA-MB-435 was re-expressed by treatment with AZA + TSA. The proliferation assay analysis showed AZA + TSA suppressed the proliferation of MDA-MB-435 cells, which were further suppressed by addition of 4-OH Tamoxifen (4-OHT). On the contrary, the proliferation of cells treated with 4-OHT alone showed no difference compared with the vehicle control. Cell cycle analysis showed AZA + TSA treated cells showed S phase arrest, which was partially attenuated by addition of estradiol (E2); furthermore, the effect of E2 on stimulation of cell cycle could be reversed by 4-OHT in the treated cells with induced ER alpha. In vivo experiment xenograft volume of MDA-MB-435 cells treated with AZA + TSA was smaller than that of the control (P < 0.01), and the xenograft of AZA + TSA treated cells was further suppressed by ovariectomy (P < 0.01).. Our data indicate that DNMT1 inhibitor AZA and HDAC inhibitor TSA play important roles in restoring sensitivity of the ER alpha negative breast cancer cells to endocrine therapy in vitro and in vivo.

Topics: Animals; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Female; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tamoxifen; Trefoil Factor-1; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

In breast cancer, approximately one-third of tumors express neither the estrogen receptor (ERalpha) nor estrogen-regulated genes such as the progesterone receptor gene (PR). Our study provides new insights into the mechanism allowing hormone-activated expression of ERalpha target genes silenced in ERalpha-negative mammary tumor cells. In cell lines derived from ERalpha-negative MDA-MB231 cells, stable expression of different levels of ERalpha from a transgene did not result in transcription of PR. A quantitative comparative analysis demonstrates that inhibiting DNA methyltransferases using 5-aza-2'-deoxycytidine or specific disruption of DNMT1 by small interfering RNAs and treatment with the histone-deacetylase inhibitor trichostatin A enabled ERalpha-mediated hormone-dependent expression of endogenous PR. We show that demethylation of a CpG island located in the first exon of PR was a prerequisite for ERalpha binding to these regulatory sequences. Although not a general requirement, DNA demethylation is also necessary for derepression of a subset of ERalpha target genes involved in tumorigenesis. PR transcription did not subsist 4 days after removal of the DNA methyltransferase blocking agents, suggesting that hormone-induced expression of ERalpha target genes in ERalpha-negative tumor cells is transient. Our observations support a model where an epigenetic mark confers stable silencing by precluding ERalpha access to promoters.

Topics: Breast Neoplasms; Cell Line, Tumor; CpG Islands; Cytidine Monophosphate; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Inhibitors; Epigenesis, Genetic; Estradiol; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Receptors, Progesterone; RNA, Small Interfering

We have previously reported on the relevance of the prevalence of CD44(+)/CD24(-/low) cells in primary breast tumors. To study regulation of CD24, we queried a number of publicly available expression array studies in breast cancer cells and found that CD24 was downregulated upon estrogen treatment. We confirmed this estrogen-mediated repression of CD24 mRNA by quantitative real-time PCR in MCF7, T47D and ZR75-1 cells. Repression was also seen at the protein level as measured by flow cytometry. CD24 was not downregulated in the ER alpha negative MDA-MB-231 cells suggesting that ER alpha was necessary. This was further confirmed by ER alpha silencing in MCF7 cells resulting in increased CD24 levels and by reintroduction of ER alpha into C4-12 cells resulting in decreased CD24 levels. Estrogen treatment did not alter half-life of CD24 mRNA and new protein synthesis was not essential for repression, suggesting a primary transcriptional effect. Histone deacetylase inhibition by Trichostatin A completely abolished the repression, but decrease of the ER alpha corepressors NCoR, LCoR, RIP140, silencing mediator of retinoid and thyroid hormone receptors, SAFB1 and SAFB2 by siRNA or overexpression of SAFB2, NCoR and silencing mediator of retinoid and thyroid hormone receptors had no effect. In silico promoter analyses led to the identification of two estrogen responsive elements in the CD24 promoter, one of which was able to bind ER alpha as shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Together, our results show that CD24 is repressed by estrogen and that this repression is a direct transcriptional effect depending on ER alpha and histone deacetylases.

Topics: Breast Neoplasms; CD24 Antigen; Cell Line, Tumor; Chromatin Immunoprecipitation; Down-Regulation; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Estrogen Receptor alpha; Estrogens; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger; Transcription, Genetic

ARHI is a maternally imprinted tumor suppressor gene that is expressed in normal breast and ovarian epithelial cells but not in most breast and ovarian cancers. Our earlier studies showed that histone deacetylases (HDACs) in complexes with transcription factors E2F1 and E2F4 play an important role in downregulating ARHI expression in breast cancer cells. To determine which HDAC or HDACs are responsible for repressing ARHI, we cotransfected vectors expressing HDACs 1-11 with an ARHI/luciferase reporter into SKBr3 and MCF-7 breast cancer cells. Expression of multiple HDACs consistently reduced ARHI promoter activity in a dose-dependent manner. We also found that the expression level of HDACs 1-3 was higher in breast cancer cell lines than in normal breast epithelial cells. In agreement with their repressive function, depletion of HDACs 1, 3 and 11 not only significantly increased the ARHI promoter activity of the transfected reporter but also activated the transcription of the endogenous ARHI gene. Furthermore, depletion or inhibition of HDACs by small interfering RNA of HDAC11 or by trichostatin A, respectively, increased E2F acetylation. Chromatin immunoprecipitation assays revealed that HDACs 1 and 3 are bound to the ARHI promoter. Taken together, our results suggest that the activity of multiple HDACs contributes to the repression of the ARHI tumor suppressor gene in breast cancer cells. Since HDAC inhibitors are now being used to treat breast cancer, the reactivation of ARHI in these cancer cells may serve as a new biomarker with which to monitor the treatment effects.

Topics: Acetylation; Breast; Breast Neoplasms; Cell Line; Cell Nucleus; Chromatin Immunoprecipitation; DNA Methylation; E2F1 Transcription Factor; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Luciferases; Ovarian Neoplasms; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; RNA, Small Interfering; Transcription, Genetic

DNA methylation and chromatin structure are two modes of epigenetic control of genome function. Although it is now well established that chromatin silencing could lead to DNA methylation, the relation between chromatin activation and DNA demethylation is unclear. It was generally believed that expression of methylated genes could only be restored by demethylating agents, such as 5-aza-deoxycytidine (5-azaCdR), and that inhibition of histone deacetylation by Trichostatin A (TSA) only activates transcription of unmethylated genes. In this report, we show that increase of histone acetylation by TSA was associated with a significant decrease in global methylation. This global demethylation occurs even when DNA replication is blocked by hydroxyurea, supporting a replication-independent-mechanism of demethylation. TSA also induces histone acetylation, demethylation and expression of the methylated E-CADHERIN and RARbeta2 genes. However, the genome-wide demethylation induced by TSA does not affect all methylated tumor suppressor genes equally suggesting that induction of acetylation and demethylation by TSA shows some gene selectivity. Taken together, our data provide evidence for a reversible crosstalk between histone acetylation and DNA demethylation, which has significant implications on the use of HDAC inhibitors as therapeutic agents.

Topics: 5' Flanking Region; Acetylation; Breast Neoplasms; Cadherins; DNA Methylation; DNA Replication; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Interspersed Repetitive Sequences; Transcription Initiation Site; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Carnitine palmitoyl transferase I (CPT1) catalyzes the transport of long-chain fatty acids into mitochondria for beta-oxidation. A link between CPT1 and apoptosis has been suggested on the basis of several experimental data. Nevertheless, results are contradictory about the effective role of CPT1 in cell survival control and cancer development. Conversely, Fatty acid synthase (FAS) enzyme, required for the synthesis of fatty acids, is found over-expressed in tumors and inhibition of FAS triggers apoptosis in human cancer cells. We have studied the tumor-specific modulation of CPT1 and FAS in human colorectal cancer (n = 11) and breast carcinomas (n = 24). CPT1 was significantly decreased in the cytoplasm of tumoral samples (p < or = 0.04), whereas FAS was increased (p < or = 0.04). A striking CPT1 nuclear localization was evident in the tumors (p < or = 0.04). In the nuclear environment the protein would modulate the levels of acetyl/acyl-CoA implicated in the regulation of gene transcription. At this purpose, we performed in vitro experiments using epithelial neoplastic (MCF-7, Caco-2, HepG2 cells) and non neoplastic cell lines (MCF-12F) confirming a nuclear localization of CPT1 protein exclusively in neoplastic cells. Moreover histone deacetylase (HDAC) activity showed significantly higher levels in nuclear extracts from neoplastic than from control cells. HDAC1 and CPT1 proteins coimmunoprecipitated in nuclear extracts from MCF-7 cells. The treatment with HDAC inhibitors such as trichostatin A and butyrate significantly decreased nuclear expression of CPT1 and its bond to HDAC1. We also identified the existence of CPT1A mRNA transcript variant 2 in MCF-7, beside to the classic isoform 1. The peculiar localization of CPT1 in the nuclei of human carcinomas and the disclosed functional link between nuclear CPT1 and HDAC1 propose a new role of CPT1 in the histonic acetylation level of tumors.

Topics: Acetylation; Adenocarcinoma; Aged; Breast Neoplasms; Carnitine O-Palmitoyltransferase; Cell Nucleus; Chromatin Immunoprecipitation; Colorectal Neoplasms; Fatty Acid Synthases; Female; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunohistochemistry; Lysine; Male; Transcription, Genetic

ARHI is a maternally imprinted tumor suppressor gene whose expression is markedly downregulated in breast cancer. Reactivation of ARHI expression in breast cancer cells is associated with increased histone H3 acetylation and decreased lysine 9 methylation of histone H3. An ARHI promoter segment that spanned bases -420 to +58 (designated the P2 region) exhibits significantly higher promoter activity in normal cells than in cancer cells. To better understand the molecular mechanisms contributing to this differential transcriptional activity, we sought to identify transcription factors that bind to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies can supershift the A2-protein complexes, whereas anti-E2F2 and 6 antibodies cannot, demonstrating that the A2 fragment interacts with specific members of the E2F family proteins. When compared with normal breast epithelial cells, breast cancer cells have significantly elevated expression of E2F1, 4 and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3 cells were cotransfected with an ARHI/luciferase reporter and E2F-expression vectors, E2F1 and 4 reduced ARHI promoter activity 2-3-fold, and this reduction could be reversed by TSA treatment. The negative regulation by E2F-HDAC complexes could also be reduced by small interfering RNA of E2F1 and 4. While the retinoblastoma protein, pRB, alone had no effect on ARHI promoter activity, repression by E2F1, but not E2F4, was enhanced by the coexpression of pRB. Taken together, our results suggest that E2F1, 4 and their complexes with HDAC play an important role in downregulating the expression of the tumor suppressor gene ARHI in breast cancer cells.

Topics: Acetylation; Binding Sites; Bone Neoplasms; Breast Neoplasms; Cell Nucleus; Cells, Cultured; Chromatin Immunoprecipitation; E2F1 Transcription Factor; E2F2 Transcription Factor; E2F4 Transcription Factor; E2F6 Transcription Factor; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Luciferases; Mammary Glands, Human; Osteosarcoma; Promoter Regions, Genetic; Response Elements; Retinoblastoma Protein; rho GTP-Binding Proteins; RNA, Small Interfering

In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

Topics: Actins; Animals; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Mice; Prostate; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; RNA, Ribosomal, 18S

Expression of Secreted frizzled related protein 1 (SFRP1), a recently identified tumor suppressor gene encoding a WNT signaling antagonist, has been found to be frequently down-regulated in breast cancer and is associated with disease progression and poor prognosis. Here, we investigated the role of epigenetic silencing of SFRP1 in breast cancer cell lines and primary breast tumors. Through analyses by methylation-specific PCR and bisulfite sequencing, promoter methylation of SFRP1 was detected in 88% (7/8) of breast cancer cell lines, 17% (1/6) of grade 1 of ductal carcinoma in situ (DCIS), 69% (9/13) of grade 2 and 3 of DCIS, 68% (19/28) of invasive ductal carcinomas (IDC) and 33% (6/18) of lobular carcinomas but not in any (0/14) of normal mammoplasty specimens and mammary epithelial organoids examined. Real-time RT-PCR studies indicated that loss or downregulation of SFRP1 expression in tumors is frequently associated with promoter hypermethylation. In addition, breast cancer cell lines with SFRP1 promoter hypermethylation reexpressed SFRP1 mRNA after treatment with 5-azaC, implying that DNA methylation is the predominant epigenetic mechanism for SFRP1 gene silencing. These findings suggest that frequent downregulation of SFRP1 expression in breast cancer can be attributed, in large part, to aberrant promoter hypermethylation in conjunction with or without histone deacetylation. Based on the frequency of tumor-specific hypermethylation in this gene, SFRP1 could provide a valuable marker for breast cancer.

Topics: Acetylation; Azacitidine; Breast; Breast Neoplasms; Cell Line; Cell Line, Tumor; Decitabine; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Genes, Tumor Suppressor; Histones; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA

Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3beta phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA) induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1) transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus.. Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3beta-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3beta/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells.. We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3beta-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.

Topics: Acetylcysteine; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Cytoplasm; Enzyme Inhibitors; Exportin 1 Protein; Fatty Acids, Unsaturated; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Karyopherins; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; RNA Interference; Transfection; Ubiquitin

Recently, we have found dramatic overexpression of ecto-5'-nucleotidase (or CD73), a glycosylphosphatidylinositol-anchored component of lipid rafts, in estrogen receptor-negative [ER-] breast cancer cell lines and in clinical samples. To find out whether there is a more general shift in expression profile of membrane proteins, we undertook an investigation on the expression of selected membrane and cytoskeletal proteins in aggressive and metastatic breast cancer cells. Our analysis revealed a remarkably uniform shift in expression of a broad range of membrane, cytoskeletal, and signaling proteins in ER- cells. A similar change was found in two in vitro models of transition to ER- breast cancer: drug-resistant Adr2 and c-Jun-transformed clones of MCF-7 cells. Interestingly, similar expression pattern was observed in normal fibroblasts, suggesting the commonality of membrane determinants of invasive cancer cells with normal mesenchymal phenotype. Because a number of investigated proteins are components of lipid rafts, our results suggest that there is a major remodeling of lipid rafts and underlying cytoskeleton in ER- breast cancer. To test whether this broadly defined ER- phenotype could be reversed by treatment with differentiating agent, we treated ER- cells with trichostatin A, an inhibitor of histone deacetylase, and observed reversal of mesenchymal and reappearance of epithelial markers. Changes in gene and protein expression also included increased capacity to generate adenosine and altered expression profile of adenosine receptors. Thus, our results suggest that during transition to invasive breast cancer there is a significant structural reorganization of lipid rafts and underlying cytoskeleton that is reversed upon histone deacetylase inhibition.

Topics: Breast Neoplasms; Cytoskeletal Proteins; Enzyme Inhibitors; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Membrane Microdomains; Membrane Proteins; Mutation; Neoplasm Invasiveness; Neoplasm Proteins; Receptors, Estrogen

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Doxorubicin; Drug Synergism; Drug Therapy, Combination; Enzyme Activation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Luciferases; Male; Mice; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We hypothesized that deregulated corepressor actions, with associated histone deacetylation activity, epigenetically suppressed vitamin D receptor (VDR) responsiveness and drives resistance towards 1alpha,25-dihydroxyvitamin D(3).. Profiling, transcriptional, and proliferation assays were undertaken in 1alpha,25(OH)(2)D(3)-sensitive MCF-12A nonmalignant breast epithelial cells, a panel of breast cancer cell lines, and a cohort of primary breast cancer tumors (n = 21).. Elevated NCoR1 mRNA levels correlated with suppressed regulation of VDR target genes and the ability of cells to undergo arrest in G(1) of the cell cycle. A similar increased ratio of corepressor mRNA to VDR occurred in matched primary tumor and normal cells, noticeably in estrogen receptor alpha-negative (n = 7) tumors. 1alpha,25(OH)(2)D(3) resistance in cancer cell lines was targeted by cotreatments with either 1alpha,25(OH)(2)D(3) or a metabolically stable analogue (RO-26-2198) in combination with either trichostatin A (TSA; histone deacetylation inhibitor) or 5-aza-2'-deoxycytidine (DNA methyltransferase inhibitor). Combinations of vitamin D(3) compounds with TSA restored VDR antiproliferative signaling (target gene regulation, cell cycle arrest, and antiproliferative effects in liquid culture) to levels which were indistinguishable from MCF-12A cells.. Increased NCoR1 mRNA is a novel molecular lesion in breast cancer cells, which acts to suppress responsiveness of VDR target genes, resulting in 1alpha,25(OH)(2)D(3) resistance and seems to be particularly associated with estrogen receptor negativity. This lesion provides a novel molecular diagnostic and can be targeted by combinations of vitamin D(3) compounds and low doses of TSA.

Topics: Azacitidine; Breast Neoplasms; Calcitriol; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cholecalciferol; Decitabine; Female; Humans; Hydroxamic Acids; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Receptors, Calcitriol; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Structure-Activity Relationship; Time Factors; Tumor Cells, Cultured

Breast tumors expressing estrogen receptor-alpha (ER) respond well to therapeutic strategies using selective ER modulators, such as tamoxifen. However, approximately 30% of invasive breast cancers are hormone independent because they lack ER expression due to hypermethylation of ER promoter. Treatment of ER-negative breast cancer cells with demethylating agents [5-aza-2'-deoxycytidine (5-aza-dC)] and histone deacetylase (HDAC) inhibitors (trichostatin A) leads to expression of ER mRNA and functional protein. Here, we examined whether epigenetically reactivated ER is a target for tamoxifen therapy. Following treatment with trichostatin A and 5-aza-dC, the formerly unresponsive ER-negative MDA-MB-231 breast cancer cells became responsive to tamoxifen. Tamoxifen-mediated inhibition of cell growth in these cells is mediated at least in part by the tamoxifen-bound ER. Tamoxifen-bound reactivated ER induces transcriptional repression at estrogen-responsive genes by ordered recruitment of multiple distinct chromatin-modifying complexes. Using chromatin immunoprecipitation, we show recruitment of two different corepressor complexes to ER-responsive promoters in a mutually exclusive and sequential manner: the nuclear receptor corepressor-HDAC3 complex followed by nucleosome remodeling and histone deacetylation complex. The mechanistic insight provided by this study might help in designing therapeutic strategies directed toward epigenetic mechanisms in the prevention or treatment of breast cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Decitabine; DNA (Cytosine-5-)-Methyltransferases; Drug Synergism; Estrogen Antagonists; Estrogen Receptor alpha; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Mi-2 Nucleosome Remodeling and Deacetylase Complex; Promoter Regions, Genetic; Repressor Proteins; RNA, Messenger; Sin3 Histone Deacetylase and Corepressor Complex; Tamoxifen

Human kallikrein 6 (KLK6) was identified based on its transient upregulation in a primary breast tumor and its subsequent silencing in a metastatic tumor from the same patient. The molecular mechanism(s) underlying the deregulated expression of KLK6 during cancer progression are currently unknown. Here, we provide evidence that aberrant expression of KLK6 is regulated at the level of transcription by multiple cooperating mechanisms. KLK6 can be reactivated in non-expressing breast cancer cells by treatment with 5-aza-2'-deoxycytidine (5-aza-dC), a compound causing DNA demethylation. Trichostatin A (TSA), an inhibitor of histone deacetylases, resulted in moderate induction of KLK6 only in MDA-MB-231 cells. However, combined 5-aza-dC/TSA treatment resulted in synergistic activation of KLK6. We show that KLK6 inactivation is associated with hypermethylation of specific CpG dinucleotides located in the KLK6 proximal promoter and overexpression with complete demethylation. These results indicate a causal role of DNA methylation and chromatin structure in cancer-associated loss of KLK6 expression. In some breast cancer cell lines, KLK6 expression could be restored by the vitamin D3 analog EB1089. Our data indicate that transcriptional deregulation of KLK6 in cancer cells during breast cancer progression is complex and certainly not uniform in different tumors, involving epigenetic mechanisms as well as pathways regulated by nuclear receptors. This allows for the pharmacological modulation of KLK6 with potential therapeutic implications.

Topics: Antineoplastic Agents; Azacitidine; Breast Neoplasms; Calcitriol; Cell Line, Tumor; Decitabine; Disease Progression; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Kallikreins; Promoter Regions, Genetic; Transcription, Genetic

A tumor suppressor function has been attributed to RUNX3, a member of the RUNX family of transcription factors. Here, we examined alterations in the expression of three members, RUNX1, RUNX2, and RUNX3, and their interacting partner, CBF-beta, in breast cancer. Among them, RUNX3 was consistently underexpressed in breast cancer cell lines and primary tumors. Fifty percent of the breast cancer cell lines (n = 19) showed hypermethylation at the promoter region and displayed significantly lower levels of RUNX3 mRNA expression (P < 0.0001) and protein (P < 0.001). In primary Singaporean breast cancers, 9 of 44 specimens showed undetectable levels of RUNX3 by immunohistochemistry. In 35 of 44 tumors, however, low levels of RUNX3 protein were present. Remarkably, in each case, protein was mislocalized to the cytoplasm. In primary tumors, hypermethylation of RUNX3 was observed in 23 of 44 cases (52%) and was undetectable in matched adjacent normal breast epithelium. Mislocalization of the protein, with or without methylation, seems to account for RUNX3 inactivation in the vast majority of the tumors. In in vitro and in vivo assays, RUNX3 behaved as a growth suppressor in breast cancer cells. Stable expression of RUNX3 in MDA-MB-231 breast cancer cells led to a more cuboidal phenotype, significantly reduced invasiveness in Matrigel invasion assays, and suppressed tumor formation in immunodeficient mice. This study provides biological and mechanistic insights into RUNX3 as the key member of the family that plays a role in breast cancer. Frequent protein mislocalization and methylation could render RUNX3 a valuable marker for early detection and risk assessment.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Core Binding Factor Alpha 3 Subunit; Decitabine; DNA Methylation; Gene Expression Regulation, Neoplastic; Gene Targeting; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Promoter Regions, Genetic

Histone deacetylase inhibitors (HDACIs) have been shown to induce apoptotic and autophagic cell death in vitro and in vivo. The molecular mechanisms that underlie these cytotoxic effects are not yet clearly understood. Recently, HDACIs were shown to induce Akt dephosphorylation by disrupting HDAC-protein phosphatase 1 (PP1) complexes. This disruption results in the increased association of PP1 with Akt, resulting in the dephosphorylation and consequent inactivation of the kinase. Akt enhances cellular survival through the phosphorylation-dependent inhibition of several pro-apoptotic proteins. Akt is an important negative regulator of GSK3beta, a kinase that has been shown to regulate apoptosis in response to various stimuli. In the present study, we investigated the role of GSK3beta in mediating the cytotoxic effects in MCF-7 breast cancer cells treated with trichostatin A (TSA), a prototype HDACI. We show that TSA induces Akt dephosphorylation in a PP1-dependent manner, resulting in activation of GSK3beta in MCF-7 cells. Similarly, knockdown of HDAC1 and-2 by small interfering RNA (siRNA) resulted in the dephosphorylation of Akt and GSK3beta. Selective inhibition of GSK3beta attenuated TSA induced cytotoxicity and resulted in enhanced proliferation following drug removal. Our findings identify GSK3beta as an important mediator of TSA-induced cytotoxicity in MCF-7 breast cancer cells.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Proto-Oncogene Proteins c-akt

To investigate the expression of ER alpha in chemically induced, ER alpha-negative human breast cancer MDA-MB-435 cells and its restoration of the responsiveness to endocrine therapy.. MDA-MB-435 cells were treated with HDAC inhibitor trichostatin A(TSA)and DNMT1 inhibitor 5-AZA-CdR (AZA). The mRNA level of ER alpha, PR and PS2 in treated MDA-MB-435 cells was detected by RT-PCR. The WST-8 (water-soluble tetrazolium salt-8) method was used to analyze the proliferation rate of the cells. Xenograft in female nude mice was used to further explore the change of proliferation rate of treated MDA-MB-435 cells in vivo.. After treatment with AZA and TSA, mRNA expression of ER alpha, PR and pS2 was up-regulated in MDA-MB-435 cells. The mRNA level of ER alpha was the hightest when MDA-MB-435 cells were treated with 2.5 micromol/L AZA and 100 ng/ml TSA. The treated MDA-MB-435 cells showed different proliferation rate in various media containing different concentration of estrodial. The MDA-MB-435 cells showed down-regulated proliferation rate after treatment with the combination of 2.5 micromol/L AZA and 100 ng/ml TSA, and 4-OH tamoxifen could suppress the growth rate of the induced MD-MBA-435 cells but not the untreated cells. The treated MDA-MB-435 cells showed slower proliferation rate than that of untreated cells in vivo (P <0. 01), and the proliferation rate of the treated MDA-MB-435 cells became lower when the nude mice were deprived of estrogen by castration (P <0. 01).. After treatment with TSA and AZA, ER alpha-negative MDA-MB-435 cells can express functional ER alpha and regain responsiveness to estrogen both in vitro and in vivo. HDAC inhibitor and DNMT1 inhibitor may play an important role in restoration of sensitivity of ER alpha-negative breast cancers to endocrine therapy.

Topics: Animals; Azacitidine; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Decitabine; DNA Modification Methylases; Enzyme Inhibitors; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Ovariectomy; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trefoil Factor-1; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays

We used the estrogen-responsive MCF-7 breast cancer cell line as a relevant model to study the anti-proliferative effects of ICI182,780 and identified the negative cell cycle regulator p21Waf1 as a specific target of ICI182,780. Furthermore, silencing of the p21Waf1 expression by small interfering RNA overcame the G0/G1 cell cycle arrest induced by ICI182,780, suggesting that the induction of p21Waf1 expression has a direct role in mediating the ICI182,780-induced G0/G1 arrest. We further demonstrated that the induction of p21Waf1 by ICI182,780 is mediated at transcriptional and gene promoter levels through the proximal Sp1 sites located near the transcription start site. Co-immunoprecipitation, DNA "pull-down," and chromatin immunoprecipitation experiments together showed that in cycling cells, estrogen receptor alpha and histone deacetylase 1 (HDAC1) are recruited to the proximal Sp1 sites of the promoter to repress p21Waf1 expression. In the presence of ICI182,780, estrogen receptor alpha and HDACs are dissociated from Sp1, resulting in increased histone acetylation and de-repression of the p21Waf1 promoter and induction of p21Waf1 expression. The fact that p21Waf1 expression is normally repressed by HDAC activity in cycling cells is further demonstrated by the finding that p21Waf1 transcription can be induced by the silencing of HDACs with small interfering RNA or treatment with HDAC inhibitors.

Topics: Antineoplastic Agents, Hormonal; Binding Sites; Breast Neoplasms; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; Estradiol; Estrogen Receptor alpha; Fulvestrant; G1 Phase; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Promoter Regions, Genetic; RNA, Small Interfering; Sp1 Transcription Factor; Transcriptional Activation; Up-Regulation

The human progesterone receptor (PR) contains multiple Ser-Pro phosphorylation sites that are potential substrates for cyclin-dependent kinases, suggesting that PR activity might be regulated during the cell cycle. Using T47D breast cancer cells stably transfected with an mouse mammary tumor virus (MMTV) chloramphenicol acetyltransferase reporter (Cat0) synchronized in different phases of the cell cycle, we found that PR function and phosphorylation is remarkably cell cycle dependent, with the highest activity in S phase. Although PR expression was reduced in the G2/M phase, the activity per molecule of receptor was markedly reduced in both G1 and G2/M phases compared to the results seen with the S phase of the cell cycle. Although PR is recruited to the MMTV promoter equivalently in the G1 and S phases, recruitment of SRC-1, SRC-3, and, consequently, CBP is reduced in G1 phase despite comparable expression levels of SRC-1 and SRC-3. In G2/M phase, site-specific phosphorylation of PR at Ser162 and at Ser294, a site previously reported to be critical for transcriptional activity and receptor turnover, was abolished. Treatment with the histone deacetylase inhibitor trichostatin A elevated G1 and G2/M activity to that of the S phase, indicating that the failure to recruit sufficient levels of active histone acetyltransferase is the primary defect in PR-mediated transactivation.

Topics: Acetyltransferases; Animals; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Nucleus; Chloramphenicol O-Acetyltransferase; Chromatin Immunoprecipitation; Cyclin-Dependent Kinases; Cytoplasm; Enzyme Inhibitors; Genes, Reporter; Histone Acetyltransferases; Humans; Hydroxamic Acids; Mammary Tumor Virus, Mouse; Mice; Phosphorylation; Promegestone; Promoter Regions, Genetic; Receptors, Progesterone; Trans-Activators; Transcription, Genetic; Transcriptional Activation

The histone deacetylase inhibitors sodium butyrate (NaBu) and trichostatin A (TSA) exhibit anti-proliferative activity by causing cell cycle arrest and apoptosis. The mechanisms by which NaBu and TSA cause apoptosis and cell cycle arrest are not yet completely clarified, although these agents are known to modulate the expression of several genes including cell-cycle- and apoptosis-related genes. The enzymes involved in the process of translation have important roles in controlling cell growth and apoptosis, and several of these translation factors have been described as having a causal role in the development of cancer. The expression patterns of the translation mechanism, namely of the elongation factors eEF1A1 and eEF1A2, and of the termination factors eRF1 and eRF3, were studied in the breast cancer cell line MCF-7 by real-time quantitative reverse transcription-polymerase chain reaction after a 24-h treatment with NaBu and TSA. NaBu induced inhibition of translation factors' transcription, whereas TSA caused an increase in mRNA levels. Thus, these two agents may modulate the expression of translation factors through different pathways. We propose that the inhibition caused by NaBu may, in part, be responsible for the cell cycle arrest and apoptosis induced by this agent in MCF-7 cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Butyrates; Cell Cycle; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Peptide Chain Elongation, Translational; Protein Biosynthesis; Tumor Cells, Cultured

Valproate (VPA) and trichostatin A (TSA), inhibitors of zinc-dependent deacetylase activity, induce reduction in the levels of mRNA encoding oestrogen receptor-alpha (ERalpha), resulting in subsequent clearance of ERalpha protein from breast and ovarian cell lines. Inhibition of oestrogen signalling may account for the endocrine disorders, menstrual abnormalities, osteoporosis and weight gain that occur in a proportion of women treated with VPA for epilepsy or for bipolar mood disorder. Transcriptome profiling revealed that VPA and TSA also modulate the expression of, among others, key regulatory components of the cell cycle. Meta-analysis of genes directly responsive to oestrogen indicates that VPA and TSA have a generally antioestrogenic profile in ERalpha positive cells. Concomitant treatment with cycloheximide prevented most of these changes in gene expression, including downregulation of ERalpha mRNA, indicating that a limited number of genes signal a hyperacetylated state within cells. Three members of the NAD-dependent deacetylases, the sirtuins, are upregulated by VPA and by TSA and sirtuin activity contributes to loss of ERalpha expression. However, prolonged inhibition of the sirtuins by sirtinol also induces loss of ERalpha from cells. Mechanistically, we show that VPA invokes reversible promoter shutoff of the ERalpha, pS2 and cyclin D1 promoters, by inducing recruitment of methyl cytosine binding protein 2 (MeCP2) with concomitant exclusion of the maintenance methylase DNMT1. Furthermore, we demonstrate that, in the presence of VPA, local DNA methylation, deacetylation and demethylation of activated histones and recruitment of inhibitory complexes occurs on the pS2 promoter.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; DNA Primers; Enzyme Inhibitors; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genetic Markers; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kinetics; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger; RNA, Neoplasm; Tamoxifen; Transcription, Genetic; Valproic Acid

In breast cancer, radiation has a central role in the treatment of brain metastasis, although tumor sensitivity might be limited. The tumor cell defense response to ionizing radiation involves activation of cell cycle checkpoint signaling. Histone deacetylase (HDAC) inhibitors, agents that cause hyperacetylation of histone proteins and thereby aberrations in the chromatin structure, may also override the DNA damage defense response and facilitate the radiation-induced mitotic cell death. In experimental metastasis models, the human breast carcinoma cell line MA-11 invariably disseminates to the central nervous system. We compared profiles of in vitro MA-11 cell cycle response to ionizing radiation and HDAC inhibition. After radiation exposure, the G2-M phase accumulation and the preceding repression of the G2 phase regulatory factors Polo-like kinase-1 and cyclin B1 required intact G2 checkpoint signaling through the checkpoint kinase CHK1, whereas the similar phenotypic changes observed with HDAC inhibition did not. MA-11 cells did not show radiation-induced expression of the G1 cell cycle inhibitor p21, indicative of a defective G1 checkpoint and consistent with a point mutation detected in the tumor suppressor TP53 gene. Increase in the p21 level, however, was observed with HDAC inhibition. Following pretreatment with the HDAC inhibitor, the efficiency of clonogenic regrowth after irradiation was reduced, which is in accordance with the concept of increased probability of mitotic cell death when the chromatin structure is disrupted. Among molecular cell cycle-targeted drugs currently in the pipeline for testing in early-phase clinical trials, HDAC inhibitors may have therapeutic potential as radiosensitizers.

Topics: Acetylation; Apoptosis; Breast Neoplasms; Cell Cycle; Cell Cycle Proteins; Checkpoint Kinase 1; Female; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Poly(ADP-ribose) Polymerases; Protein Kinases; Radiation-Sensitizing Agents; Radiation, Ionizing; Signal Transduction; Tumor Suppressor Protein p53

To explore the molecular mechanisms for the similarities between inherited and noninherited forms of breast cancer, we tested the hypothesis that inactivation of BRCA1 by promoter hypermethylation is associated with reduced gene copy number and chromosome 17 aneusomy as observed in tumors from BRCA1 mutation carriers. Using a combination of methylation-specific PCR analysis and fluorescence in situ hybridization, we observed varying degrees of promoter methylation in 39 of 131 (29.8%) primary tumors. Despite significant tumor heterogeneity, mean copy numbers of BRCA1 and CEP17 per cell were lower in methylated cases compared with unmethylated cases [1.78 versus 2.30 (P = 0.001) and 1.85 versus 2.29 (P = 0.005), respectively]. Methylation was more frequently observed in younger women (P = 0.05) with high-grade (P = 0.001), estrogen receptor-negative (P = 0.04), and progesterone receptor-negative (P = 0.01) tumors. Moreover, methylation was associated with reduced or absent BRCA1 transcripts, which was reversible in the heavily BRCA1-methylated cell line UACC3199 following treatment with 5-aza-2'-deoxycytidine and trichostatin A. We identified five CpGs at positions -533, -355, -173, -21, and +44 as critical in the reexpression of BRCA1. We conclude that BRCA1 methylation contributes to a subset of sporadic breast cancers with the resulting molecular and clinicopathologic phenotype similar to that of hereditary BRCA1-associated breast cancers. Our data support a model of carcinogenesis in which BRCA1 promoter methylation may serve as a "first hit," much like an inherited germ line mutation, and promote tumor progression down a restricted set of molecular pathways.

Topics: Aneuploidy; Antimetabolites, Antineoplastic; Azacitidine; BRCA1 Protein; Breast; Breast Neoplasms; Cell Line, Tumor; Chromosomes, Human, Pair 17; Decitabine; DNA Methylation; Female; Gene Dosage; Gene Expression; Genes, BRCA1; Humans; Hydroxamic Acids; Lymphocytes; Middle Aged; Promoter Regions, Genetic; RNA, Messenger

Many cases of breast cancer show loss of estrogen receptor (ER) alpha expression, which leads to unresponsiveness to antihormonal treatment even though there is no loss of the structurally and biochemically similar ER beta. ER activity is positively and negatively regulated by transcriptional regulators such as histone deacetylase (HDAC), which is known to be a negative ER regulator. Here, we evaluated using ER beta as an alternative target for tamoxifen therapy by treating ER alpha-negative, beta-positive breast cancer cells with the HDAC inhibitor trichostatin A (TSA), and testing whether tamoxifen responsiveness increased following upregulation of ER beta. TSA enhanced the overall ER transcriptional activity in these cells, as visualized by estrogen response element-regulated reporter and the expression of progesterone receptor, a known ER target, without ER alpha restoration. Additionally, TSA induced the expression and nuclear translocation of ER beta but not alpha, suggesting that these actions leading to increase of ER transcriptional activity are mediated through ER beta rather than alpha. Furthermore, following treatment with TSA, the formerly unresponsive MDA-MB-231 and Hs578T breast cancer cells became responsive to tamoxifen. However, reduction of ER beta expression by short interfering RNA abrogated this TSA-induced sensitization effect in these cells. Together, these results show that the HDAC inhibitor TSA sensitized ER alpha-negative, antihormone-unresponsive breast cancer cells to tamoxifen treatment possibly by upregulating ER beta activity.

Topics: Active Transport, Cell Nucleus; Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Receptors, Estrogen; Receptors, Progesterone; Response Elements; RNA, Messenger; RNA, Neoplasm; Tamoxifen

Resistance to the cytotoxic actions of antineoplastic drugs, whether intrinsic or acquired, remains a barrier to the establishment of curative chemotherapy regimens for advanced breast cancer. Over-expression of P-glycoprotein (P-gp), encoded by the MDR1 gene and known to mediate resistance to many antineoplastic drugs, may contribute to poor breast cancer treatment outcome. Nonetheless, the precise molecular mechanisms responsible for high or low level P-gp expression in breast cancer cells have not been established. We assessed the role of DNA hypermethylation near the MDR1 transcriptional regulatory region in MDR1 expression in MCF-7 breast cancer cells, which fail to express MDR1 mRNA, and MCF-7/ADR cells, known to express high MDR1 mRNA levels. When compared to MCF-7/ADR cells, MCF-7 cells manifested markedly diminished MDR1 transcription rates by nuclear run-off assay, but equivalent MDR1 promoter trans-activation activity in transient transfection experiments, indicating that cis factors were most likely responsible for the differences in MDR1 transcription between MCF-7/ADR cells and MCF-7 cells. Bisulfite genomic sequencing analyses revealed substantially less extensive MDR1 promoter methylation in MCF-7/ADR cells than in MCF-7 cells, suggesting that CpG dinucleotide methylation might contribute to the observed MDR1 transcription differences. Chromatin immunoprecipitation analyses indicated an inactive MDR1 chromatin conformation in MCF-7 cells, with a paucity of acetylated histones and the presence of 5-mC-binding proteins MeCP2 and MBD2, and an active MDR1 chromatin conformation in MCF-7/ADR cells, with an abundance of acetylated histones and the presence of the transcriptional trans-activator YB-1. Stable MCF-7 sublines which had been treated with the DNA methyltransferase inhibitor 5-azacytidine, exhibited a reduction in MDR1 promoter methylation and a complex MDR1 chromatin configuration, characterized by the simultaneous presence of transcriptional activators and repressors. In this state, MDR1 expression was markedly sensitive to treatment with the histone deacetylase inhibitor trichostatin A.

Topics: Acetylation; Antibiotics, Antineoplastic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azacitidine; Breast Neoplasms; Chromatin; Chromatin Immunoprecipitation; Decitabine; DNA Methylation; DNA Modification Methylases; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; RNA, Messenger; Transcriptional Activation; Tumor Cells, Cultured

In this study, we have analysed the effects of histone deacetylase (HDAC) inhibition on estrogen receptor (ER) expression and on its transcriptional activity in response to antiestrogens. In several breast cancer cell lines, trichostatin A (TSA), a potent HDAC inhibitor, strongly decreases ERalpha expression in a dose-dependent manner. This repression is observed independently of the presence of ligand and also occurs in ovarian and endometrial cell lines. In addition, we show that in MCF7 cells bearing a stably transfected reporter plasmid (MELN cells), partial antiestrogens such as 4-OH-tamoxifen (OHTam), raloxifen or LY117018, switch to an agonist activity upon HDAC inhibition. This effect is blocked by the pure antiestrogen ICI182780 and exhibits a half-maximal concentration of OHTam equivalent to its affinity for ERalpha. The TSA-dependent decrease of ERalpha expression is required to induce the agonist switch of OHTam properties as it is lost in cells constitutively expressing exogenous receptors (MELN-ERalpha or ERbeta). By contrast, the transrepression activity of OHTam is abolished by TSA independently of the decrease of ERalpha expression. Interestingly, in MELN-ERalpha, ICI182780 remains inhibitory suggesting the involvement of HDAC-independent mechanisms. Finally, in the absence of TSA, transcriptional activity in response to OHTam is significantly raised in MELN cells expressing low levels of ERalpha after transfection of antisense oligonucleotides. In conclusion, inhibition of HDAC enzymatic activity and modulation of ERalpha levels tightly control the relative agonist activity of partial antiestrogens on a stably integrated reporter transgene.

Topics: Breast Neoplasms; Enzyme Inhibitors; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogen Receptor Modulators; Female; Fulvestrant; Genes, Reporter; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Pyrrolidines; Raloxifene Hydrochloride; Response Elements; Tamoxifen; Thiophenes; Transcription, Genetic; Tumor Cells, Cultured

The cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors. Functional studies suggest that CRBP downregulation contributes to breast tumor progression. What is the mechanism underlying CRBP downregulation in cancer? Here we investigated the hypothesis that CRBP is epigenetically silenced through DNA hypermethylation in human and mouse breast cancer.. Bisulfite sequencing of CRBP in a panel of 6 human breast cancer cell lines demonstrated that, as a rule, CRBP hypermethylation is closely and inversely related to CRBP expression and identified one exception to this rule. Treatment with 5-azacytidine, a DNA methyltransferase inhibitor, led to CRBP reexpression, supporting the hypothesis that CRBP hypermethylation is a proximal cause of CRBP silencing. In some cells CRBP reexpression was potentiated by co-treatment with retinoic acid, an inducer of CRBP, and trichostatin A, a histone deacetylase inhibitor. Southern blot analysis of a small panel of human breast cancer specimens identified one case characterized by extensive CRBP hypermethylation, in association with undetectable CRBP mRNA and protein. Bisulfite sequencing of CRBP in MMTV-Myc and MMTV-Neu/NT mammary tumor cell lines extended the rule of CRBP hypermethylation and silencing (both seen in MMTV-Myc but not MMTV-Neu/NT cells) from human to mouse breast cancer and suggested that CRBP hypermethylation is an oncogene-specific event.. CRBP hypermethylation appears to be an evolutionarily conserved and principal mechanism of CRBP silencing in breast cancer. Based on the analysis of transgenic mouse mammary tumor cells, we hypothesize that CRBP silencing in human breast cancer may be associated with a specific oncogenic signature.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; DNA Methylation; Down-Regulation; Evolution, Molecular; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Mice; Oncogenes; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

Trichostatin A, an antifungal antibiotics, and HC-toxin are potent and specific inhibitors of histone deacetylase activity. Histone deacetylase inhibitors are new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest. In this study, the antiproliferative activities of trichostatin A and HC-toxin were compared between estrogen receptor positive human breast cancer cell MCF-7 and estrogen receptor negative human breast cancer cell MDA-MB-468. Trichostatin A and HC-toxin showed potent antiproliferative activity in both MCF-7 and MDA-MB-468 cells. In MCF-7 cells that contain high level estrogen receptor, trichostatin A and HC-toxin brought about three-times more potent cell growth inhibitory effect than estrogen receptor negative MDA-MB-468 cells. Both trichostatin A and HC-toxin showed cell cycle arrest at G2/M phases of MCF-7 and MDA-MB-468 cells in a dose- and time-dependent manner. Trichostatin A and HC-toxin also induced apoptosis from MCF-7 and MDA-MB-468 cells in a dose- and time-dependent manner. Results of this study suggested that antiproliferative effects of trichostatin A and HC-toxin might be involved in estrogen receptor signaling pathway, but cell cycle arrest and apoptosis of trichostatin A and HC-toxin might not be involved in estrogen receptor system of human breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Growth Inhibitors; Humans; Hydroxamic Acids; Peptides, Cyclic; Receptors, Estrogen

Proliferation of the non-malignant breast epithelial cell line, MCF-12A, is sensitively and completely inhibited by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) (ED90 = 70 nM), We used real time RT-PCR to demonstrate that the relative resistance to 1alpha,25(OH)(2)D(3) of MDA-MB-231 cells (ED50 > 100 nM) correlated with significantly reduced Vitamin D receptor (VDR) and increased NCoR1 nuclear receptor co-repressor mRNA (0.1-fold reduction in VDR and 1.7-fold increase in NCoR1 relative to MCF-12A (P < 0.05)). This molecular lesion can be targeted by co-treating cells with 1alpha,25(OH)(2)D(3) or potent analogs and the histone deacetylation inhibitor trichostatin A (TSA). For example, the co-treatment of 1,25-dihydroxy-16,23,Z-diene-26,27-hexafluoro-19-nor Vitamin D(3) (RO-26-2198) (100 nM) plus TSA results in strong additive antiproliferative effects in MDA-MB-231 cells. This may represent novel chemotherapeutic regime for hormone insensitive breast cancer.

Topics: Breast Neoplasms; Calcitriol; Cell Division; Cell Line, Tumor; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Reverse Transcriptase Polymerase Chain Reaction

Histone deacetylase inhibitors are new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest. Trichostatin A, an antifungal antibiotic, and HC-toxin are potent and specific inhibitors of histone deacetylase activity. In this study, we have examined the antiproliferative activities of trichostatin A and HC-toxin in estrogen receptor positive human breast cancer, T47D cells. Both trichostatin A and HC-toxin showed potent antiproliferative efficacy and cell cycle arrest at G2/M in T47D human breast cancer cells in a dose-dependent manner. Trichostatin A caused potent apoptosis of T47D human breast cancer cells and trichostatin A-induced apoptosis might be involved in an increase of caspase-3/7 activity. HC-toxin evoked apoptosis of T47D cells and HC-toxin induced apoptosis might not be mediated through direct increase in caspase-3/7 activity. We have identified potent activities of antiproliferation, apoptosis, and cell cycle arrest of trichostatin A and HC-toxin in estrogen receptor positive human breast cancer cell line T47D.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Caspase 3; Caspase 7; Caspases; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Antibody Technique; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Peptides, Cyclic; Toxins, Biological

Estrogen receptor alpha (ERalpha)-positive breast cancer cell lines are up to 10 times more sensitive than ERalpha-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The purpose of the study was to investigate the mechanisms underlying this differential response.. In the ERalpha-positive MCF-7 cell line, TSA repressed ERalpha and cyclin D1 transcription and induced ubiquitin dependent proteasomal degradation of cyclin D1, leading primarily to G(1)-S-phase cell cycle arrest. By contrast, cyclin D1 degradation was enhanced but its transcription unaffected by TSA in the ERalpha-negative MDA-MB-231 cell line, which arrested in G(2)-M phase. Cyclin D1 degradation involved Skp2/p45, a regulatory component of the Skp1/Cullin/F-box complex; silencing SKP2 gene expression by RNA interference stabilized cyclin D1 and abrogated the cyclin D1 down-regulation response to TSA.. Tamoxifen has been shown to inhibit ERalpha-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ERalpha-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ERalpha-dependent and ERalpha-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Cycle; Cell Proliferation; Cyclin D1; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Endopeptidases; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; RNA Interference; S-Phase Kinase-Associated Proteins; Tamoxifen; Transcription, Genetic; Tumor Cells, Cultured; Uterine Neoplasms

Absence of the estrogen receptor alpha (ER) in human breast cancer cells is an indicator of poor prognosis, and predictive of lack of response to hormonal therapy. Previous studies in our laboratory and others have shown that epigenetic regulation, including DNA methylation and histone deacetylation, are common mechanisms leading to ER gene silencing. Through the use of pharmacologic inhibitors, 5-aza 2'deoxycytidine (AZA) and Trichostatin A (TSA), we have shown that alterations in both of these mechanisms results in synergistic reexpression of ER mRNA and functional protein. These alterations may play a larger role in stimulation of cell signaling pathways leading to ER expression. We have utilized newly developed genome wide screening microarray techniques to identify gene(s) contributing to the hormone independent phenotype and AZA/TSA mediated ER expression. From this screen, we identified and confirmed expression of 4 candidate genes (PP2A, XCL1, THY1 and NBC4) as potential regulators of the hormone independent phenotype. Expression of two genes, XCL1 and PP2A, appeared to be correlated with ER expression. PP2A expression was not changed with ER degradation using ICI 182,780 whereas XCL1 expression decreased in the presence of AZA/TSA and ICI 182,780. This suggests that PP2A may be a determinant of ER expression while XCL1 appears to be ER responsive and downstream of ER expression. These gene products may be novel targets to be further explored in the development of new therapeutics for ER negative breast cancer.

Topics: Acetylation; Azacitidine; Breast Neoplasms; Chemokines, C; Decitabine; DNA Methylation; DNA Modification Methylases; Epigenesis, Genetic; Estrogen Receptor alpha; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lymphokines; Oligonucleotide Array Sequence Analysis; Phosphoprotein Phosphatases; Protein Phosphatase 2; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sialoglycoproteins; Tumor Cells, Cultured

Histone deacetylase activity is potently inhibited by hydroaximc acid derivatives such as suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). These inhibitors specifically induce differentiation/apoptosis of transformed cells in vitro and suppress tumor growth in vivo. Because of its low toxicity, SAHA is currently evaluated in clinical trials for the treatment of cancer. SAHA and TSA induce apoptosis, which is characterized by mitochondrial stress, but so far, the critical elements of this apoptotic program remain poorly defined. To characterize in more detail this apoptotic program, we used human cell lines containing alterations in important elements of apoptotic response such as: p53, Bcl-2, caspase-9, and caspase-3. We demonstrate that caspase-9 is critical for apoptosis induced by SAHA and TSA and that efficient proteolytic activation of caspase-2, caspase-8, and caspase-7 strictly depends on caspase-9. Bcl-2 efficiently antagonizes cytochrome c release and apoptosis in response to both histone deacetylase inhibitors. We provide evidences that translocation into the mitochondria of the Bcl-2 family member Bid depends on caspase-9 and that this translocation is a late event during TSA-induced apoptosis. We also demonstrate that the susceptibility to TSA- and SAHA-induced cell death is regulated by p53.

Topics: Adenovirus E1A Proteins; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Breast Neoplasms; Carrier Proteins; Caspase 2; Caspase 3; Caspase 7; Caspase 8; Caspase 9; Caspases; Cytochrome c Group; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vorinostat

Acetylation of histones in chromatin is one mechanism involved in the regulation of gene transcription and is tightly controlled by the balance of acetyltransferase and deacetylase (HDAC) activities. In cancer, some genes are repressed by the inappropriate recruitment of HDACs, e.g., tumor suppressor genes. To understand the genomic effects of HDAC inhibition on gene transcription we studied the gene expression profiles of T24 bladder and MDA breast carcinoma cells treated with three HDAC inhibitors, suberoylanilide hydroxamic acid, trichostatin A, and MS-27-275. The gene expression profiles of the HDAC inhibitors were generally similar to one another and differed substantially from those produced by structurally related inactive analogues; consequently, the changes in gene expression are mechanism-based. Hierarchical clustering of expression profiles demonstrated a greater similarity between the two hydroxamate-containing inhibitors (suberoylanilide hydroxamic acid and trichostatin A) than with MS-27-275. This difference was also supported by cell phenotypic experiments. As many genes were down-regulated as up-regulated by HDAC inhibitor treatment. Comparison of the data sets defined a common ("core") set of 13 genes regulated by all of the HDAC inhibitors in three cell lines, 8 up-regulated and 5 down-regulated. Ten of 13 genes were confirmed in dose response studies in T24 cells by quantitative-PCR. The core regulated genes are involved predominantly in cell cycle/apoptosis and DNA synthesis in response to HDAC inhibitors. These data will aide in understanding the complex set of events in cells in response to chromatin remodeling induced by HDAC inhibition, which may be responsible for antitumor effects.

Topics: Benzamides; Breast Neoplasms; Chromatin; DNA Primers; DNA, Neoplasm; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Oligonucleotide Array Sequence Analysis; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Up-Regulation; Urinary Bladder Neoplasms

Androgen and progesterone receptors (AR and PR) are two determining factors in gonadal differentiation that are highly expressed in developing and mature gonads. Loss of AR results in XY sex reversal and mutations causing reduced AR activity lead to varying degrees of defects in masculinization. Female PR knockout mice are infertile due to ovarian defects. While much has been discovered about positive regulation of these receptors by coactivators little is known about repression of the transcriptional activity of AR and PR in the presence of agonists. In this study we assessed the effect of SMRT and DAX-1 on AR and PR activity in the presence of both agonists and partial antagonists. We show that SMRT and DAX-1 repress agonist-dependent activity of both receptors, and the mechanism of repression includes disruption of the receptor dimer interactions rather than recruitment of histone deacetylases. We demonstrate that endogenous agonist-bound PR and DAX-1 in T47D breast cancer cells and endogenous AR and DAX-1 in LNCaP prostate cancer cells can be coimmunoprecipitated suggesting that the interaction is physiological. Surprisingly, although DAX-1 represses partial antagonist activity of AR, it was ineffective in repressing partial antagonist induced activity of PR. In contrast to most reported repressors, the expression of DAX-1 is restricted. We found that although DAX-1 is expressed in normal human prostate, its expression is strongly reduced in benign prostatic hyperplasia suggesting that DAX-1 plays a role in limiting AR activity in prostate.

Topics: Animals; Binding Sites; Breast Neoplasms; COS Cells; DAX-1 Orphan Nuclear Receptor; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Hormone Antagonists; Humans; Hydroxamic Acids; Male; Metribolone; Mifepristone; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Co-Repressor 2; Promoter Regions, Genetic; Prostate; Prostatic Hyperplasia; Protein Structure, Tertiary; Protein Synthesis Inhibitors; Receptors, Androgen; Receptors, Calcitriol; Receptors, Interferon; Receptors, Progesterone; Receptors, Retinoic Acid; Repressor Proteins; Testosterone Congeners; Tumor Suppressor Protein p53

Sp3 transcription factor can either activate or repress target gene expression. However, the molecular event that controls this dual function is unclear. We previously reported (Ammanamanchi, S., and Brattain, M. G. (2001) J. Biol. Chem. 276, 3348-3352) that unmodified Sp3 acts as a transcriptional repressor of transforming growth factor-beta receptors in MCF-7L breast cancer cells. We now report that histone deacetylase inhibitor trichostatin A (TSA) induces acetylation of Sp3, which acts as a transcriptional activator of transforming growth factor-beta receptor type II (RII) in MCF-7L cells. Mutation analysis indicated the TSA response is mediated through a GC box located on the RII promoter, which was previously identified as an Sp1/Sp3-binding site that was critical for RII promoter activity. Ectopic Sp3 expression in Sp3-deficient MCF-7E breast cancer cells repressed RII promoter activity in the absence of TSA. However, in the TSA-treated MCF-7E cells ectopic Sp3 activated RII promoter. Histone acetyltransferase p300 was shown to acetylate Sp3. Sp3-mediated RII promoter activity was stimulated by wild type p300 but not the histone acetyltransferase domain-deleted mutant p300 in MCF-7L cells, suggesting the positive effect of p300 acetylase activity on Sp3. Consequently, the results presented in this manuscript demonstrate that acetylation acts as a switch that controls the repressor and activator role of Sp3.

Topics: Acetylation; Breast Neoplasms; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Kinetics; Multigene Family; Pancreatic Neoplasms; Pregnancy Proteins; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Repressor Proteins; Trans-Activators; Tumor Cells, Cultured

Breast and prostate cancer are leading causes of cancer death in the Western world. Hormone ablation is the primary therapy for invasive disease, but the tumour often recurs in an androgen or oestrogen receptor negative form for which novel therapies are sought urgently. The vitamin D receptor (VDR) may provide an important alternative therapeutic target. However, cancer cell line models from these tissues display a range of sensitivities to the antiproliferative effects of 1alpha,25dihydroxyvitamin D3 (1alpha,25(OH)2D3). The reason for apparent 1alpha,25(OH)2D3 insensitivity is currently unknown and we have investigated epigenetic mechanisms that may suppress the transcriptional activity of the VDR. Nuclear co-repressors have associated histone deacetylase (HDAC) activity, which keeps chromatin in a closed, transcriptionally silent state. We have found that the aggressive cancer cell lines with relative insensitivity to 1alpha,25(OH)2D3 have elevated nuclear co-repressor levels. For example, PC-3 prostate cancer cells have a significant 1.8-fold elevation in the co-repressor SMRT compared to normal epithelial cells (P < 0.05). We believe that a combination of elevated co-repressor level with reduced VDR content can cause 1alpha,25(OH)2D3 resistance. Consistent with this, we have shown that combining a low dose of HDAC inhibitor Trichostatin A (15 nM TSA) with 1alpha,25(OH)2D3 (100 nM) synergistically inhibits the proliferation of PC-3 prostate and MDA-MB-231 breast cancer cell lines. The inhibition of proliferation was potentiated further by treating cells with 19-nor-hexafluoride vitamin D3 analogues instead of 1alpha,25(OH)2D3, plus TSA. For example, the combination of 1alpha,25(OH)2D3 and TSA-inhibited MDA-MB-231 cell proliferation by 38% (+/-5%), whereas Ro26-2198 (1alpha,25-(OH)2-16,23Z-diene-26,27-F6-19-nor-D3) and TSA inhibited growth by 62% (+/-2%). Therapeutically the hypercalcaemic side effects associated with 1alpha,25(OH)2D3 could be minimized by combining low doses of potent 1a,25(OH)2D3 analogues with HDAC inhibitors as a novel anticancer regime for hormone-insensitive prostate and breast cancer.

Topics: Androgens; Antineoplastic Agents; Breast Neoplasms; Cell Division; Cholecalciferol; DNA-Binding Proteins; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ligands; Male; Neoplasms, Hormone-Dependent; Nuclear Receptor Co-Repressor 2; Prostatic Neoplasms; Repressor Proteins; Tumor Cells, Cultured; Vitamin D

The interaction between 17beta-estradiol and estrogen receptor alpha (ER-alpha) plays an important role in breast carcinogenesis and breast cancer treatment. ER-alpha is a critical growth regulatory gene in breast cancer and its expression level is tightly linked to the prognosis and treatment outcomes of breast cancer patients. Loss of ER-alpha expression in breast epithelial cells is critical for breast cancer progression. The underlying molecular mechanisms for this loss, however, are poorly defined. Histone deacetylases (HDACs) are implicated in the alteration of chromatin assembly and tumorigenesis. We show that histone deacetylase 1 (HDAC1) interacts with ER-alpha in vitro and in vivo and suppresses ER-alpha transcription activity. The interaction of HDAC1 with ER-alpha was mediated by the AF-2 and DBD domains of ER-alpha. We observed an endogenous interaction of HDAC1 with ER-alpha in breast cancer cells, which was decreased in the presence of estrogen. Interestingly, overexpression of HDAC1 in stable transfected MCF-7 clones induced loss of ER-alpha and significantly increased cell proliferation and colony formation, as compared to the control MCF-7 cells, whereas treatment of stable MCF-7 clones with the HDAC specific inhibitor trichostatin A (TSA) induced re-expression of ER-alpha mRNA and protein. Our findings strongly suggest that HDAC1 affects breast cancer progression by promoting cellular proliferation in association with a reduction in both ER-alpha protein expression and transcriptional activity. Thus, HDAC1 may be a potential target for therapeutic intervention in the treatment of a subset of ER-negative breast cancers.

Topics: Breast Neoplasms; Disease Progression; Estrogen Receptor alpha; Female; Histone Deacetylase 1; Histone Deacetylases; Humans; Hydroxamic Acids; Receptors, Estrogen; Tumor Cells, Cultured

We analysed the antiproliferative activity of various histone deacetylase (HDAC) inhibitors such as trichostatin A (TSA) on human breast cancer cells. We observed a lower sensitivity to HDAC inhibition for oestrogen receptor negative (ER-) versus positive (ER+) cell lines. This differential response was associated neither with a modification of drug efflux via the multidrug resistance system nor with a global modification of histone acetyltransferase (HAT)/HDAC activities. In contrast, we demonstrated that in ER+ breast cancer cells the p21(WAF1/CIP1) gene was more sensitive to TSA regulation and was expressed at higher levels. These differences were observed both in transient transfection experiments and on the endogenous p21(WAF1/CIP1) gene. The Sp1 transcription factor, which was shown to interact in vitro with both class I and class II HDACs, is sufficient to confer the differential sensitivity to TSA and participated in the control of p21(WAF1/CIP1) basal expression. Finally, re-expression of ERalpha following adenoviral infection of ER- breast cancer cells increased both p21(WAF1/CIP1) protein accumulation and the growth inhibitory activity of TSA. Altogether, our results highlight the key role of ERalpha and p21(WAF1/CIP1) gene expression in the sensitivity of breast cancer cells to hyperacetylating agents.

Topics: Acetylation; Antineoplastic Agents; Breast Neoplasms; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Neoplasm Proteins; Receptors, Estrogen; Sp1 Transcription Factor; Transfection; Tumor Cells, Cultured

Several anticancer drugs target DNA or enzymes acting on the DNA. Because chromatin DNA is tightly compacted, accessibility to the drug target may reduce the efficiency of these anticancer drugs. We thus treated four human cancer cell lines and two normal epithelial cell lines with either trichostatin A (TSA) or SAHA, two histone deacetylase inhibitors, before exposing the cells to VP-16, ellipticine, camptothecin, doxorubicin, cisplatin, 5-fluorouracil, or cyclophosmamide. Pretreatment with TSA or SAHA increased the killing efficiency of VP-16, ellipticine, doxorubicin, and cisplatin. The magnitude of sensitization is cell type specific and is >10-fold for VP-16 in D54, a brain tumor cell line intrinsically resistant to topoisomerase II inhibitors. Topoisomerase II levels and activity were not affected by this treatment, but p53, p21, and Gadd45 protein levels were markedly induced. Moreover, pretreatment with TSA also increased VP-16-induced apoptosis in a p53-dependent and -independent manner. Treating the cells in the reverse order (anticancer drug first, followed by TSA or SAHA) had no more cytotoxic effect than the drug alone. These data suggest that loosening-up the chromatin structure by histone acetylation can increase the efficiency of several anticancer drugs targeting DNA. This may be advantageous for treating tumors intrinsically resistant to these drugs.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; DNA Topoisomerases, Type II; DNA, Neoplasm; Drug Synergism; Enzyme Inhibitors; Etoposide; Gene Expression; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Tumor Suppressor Protein p53; Vorinostat

Urokinase-type plasminogen activator (uPA) is a member of the serine protease family and can break down various components of the extracellular matrix to promote growth, invasion, and metastasis of several malignancies including breast cancer. In the current study we examined the role that the DNA methylation machinery might be playing in regulating differential uPA gene expression in breast cancer cell lines. uPA mRNA is expressed in the highly invasive, hormone-insensitive human breast cancer cell line MDA-MB-231 but not in hormone-responsive cell line MCF-7. Using methylation-sensitive PCR, we show that 90% of CpG dinucleotides in the uPA promoter are methylated in MCF-7 cells, whereas fully demethylated CpGs were detected in MDA-MB-231 cells. uPA promoter activity, which is directly regulated by the Ets-1 transcription factor, is inhibited by methylation as determined by uPA promoter-luciferase reporter assays. We then tested whether the state of expression and methylation of the uPA promoter correlates with the global level of DNA methyltransferase and demethylase activities in these cell lines. We show that maintenance DNA methyltransferase activity is significantly higher in MCF-7 cells than in MDA-MB-231 cells, whereas demethylase activity is higher in MDA-MB-231 cells. We suggest that the combination of increased DNA methyltransferase activity with reduced demethylase activity contributes to the methylation and silencing of uPA expression in MCF-7 cells. The converse is true in MDA-MB-231 cells, which represents a late stage highly invasive breast cancer. The histone deacetylase inhibitor, Trichostatin A, induces the expression of the uPA gene in MDA-MB-231 cells but not in MCF-7 cells. This supports the hypothesis that DNA methylation is the dominant mechanism involved in the silencing of uPA gene expression. Taken together, these results provide insight into the mechanism regulating the transcription of the uPA gene in the complex multistep process of breast cancer progression.

Topics: Base Sequence; Binding Sites; Breast Neoplasms; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Molecular Sequence Data; Neoplasm Invasiveness; Promoter Regions, Genetic; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The antitumor activity of histone deacetylase (HDAC) inhibitors has been linked to gene expression induced by acetylation of histone and nonhistone proteins; but the molecular basis for their antitumor selectivity remains largely unknown. With development of a genomically integrated, ErbB2 promoter-reporting breast cancer cell screen, ErbB2 promoter inhibiting activity was observed by the HDAC inhibitors trichostatin A (TSA) and sodium butyrate. Paradoxically, these agents stimulated the episomal form of this ErbB2 promoter-reporter introduced by transient transfection. Transcriptional run-off assays in ErbB2 amplified and overexpressing breast cancer cells confirmed that within 5 h, TSA exposure profoundly inhibits ErbB2 transcript synthesis from the amplified oncogene yet preserves transcription from single copy genes such as the epithelial-specific Ets family member, ESX. Northern analyses of ErbB2-overexpressing breast cancer lines (SKBR3, BT-474, and MDA-453) showed that within 24 h of submicromolar treatment by TSA, ESX transcript levels increase while ErbB2 transcript levels rapidly decline, with no TSA effect apparent on the open chromatin configuration of either gene as monitored by DNase I hypersensitivity. Actinomycin D studies confirmed that in addition to inhibiting ErbB2 transcript synthesis, TSA selectively destabilizes mature ErbB2 transcripts enhancing their decay. Whereas TSA markedly reduced ErbB2 protein levels in these overexpressing cell lines, TSA treatment of MCF/HER2-18 cells engineered to overexpress the ErbB2 receptor under control of a heterologous promoter increased their expression of ErbB2 protein. These findings suggest that further studies are warranted to determine whether ErbB2-positive human cancers represent unusually sensitive clinical targets for HDAC inhibitor therapy.

Topics: Blotting, Western; Breast Neoplasms; Butyric Acid; Cell Survival; Deoxyribonuclease I; DNA Probes; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Luciferases; Nucleotide Mapping; Promoter Regions, Genetic; Receptor, ErbB-2; Response Elements; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Transcriptional repression of the transforming growth factor-beta (TGF-beta) type II receptor (TbetaRII) gene is one of several mechanisms leading to TGF-beta resistance. Previously, we have shown that MS-275, a synthetic inhibitor of histone deacetylase (HDAC), specifically induces the expression of the TbetaRII gene and restores the TGF-beta signaling in human breast cancer cell lines. However, little is known about the mechanism by which inhibition of HDAC activates TbetaRII expression. MS-275 treatment of cells expressing a wild-type TbetaRII promoter/luciferase construct resulted in a 10-fold induction of the promoter activity. DNA transfection and an electrophoretic mobility shift assay showed that the induction of the TbetaRII promoter by MS-275 requires the inverted CCAAT box and its cognate binding protein, NF-Y. In addition, a DNA affinity pull-down assay indicated that the PCAF protein, a transcriptional coactivator with intrinsic histone acetyltransferase (HAT) activity, is specifically recruited to the NF-Y complex in the presence of either MS-275 or trichostatin A. Based on these results, we suggest that treatment with the HDAC inhibitor induces TbetaRII promoter activity by the recruitment of the PCAF protein to the NF-Y complex, interacting with the inverted CCAAT box in the TbetaRII promoter.

Topics: Acetyltransferases; Base Sequence; Benzamides; Breast Neoplasms; CCAAT-Binding Factor; Cell Nucleus; DNA; Enzyme Inhibitors; Gene Expression Regulation; Genes, Reporter; Histone Acetyltransferases; Histone Deacetylases; Humans; Hydroxamic Acids; Luciferases; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Promoter Regions, Genetic; Protein Binding; Protein Serine-Threonine Kinases; Pyridines; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Saccharomyces cerevisiae Proteins; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Loss of expression of retinoic acid receptor beta2 (RARbeta2), a potent tumor suppressor gene, is commonly observed during breast carcinogenesis. RARbeta2 silencing can be traced to epigenetic chromatin changes affecting the RARbeta P2 promoter. Here we show that retinoic acid therapy fails to induce RARbeta2 in primary breast tumors, which carry a methylated RARbeta P2 promoter. DNA methylation leads to repressive chromatin deacetylation at RARbeta P2. By inducing an appropriate level of histone reacetylation at RARbeta P2 we could reactivate endogenous RARbeta2 transcription from unmethylated as well as methylated RARbeta P2 in breast cancer cell lines and xenograft tumors, and obtain significant growth inhibition both in vitro and in vivo. This study may have translational implications for breast cancer and other cancers carrying an epigenetically silenced RARbeta P2 promoter.

Topics: Acetylation; Antineoplastic Agents; Breast Neoplasms; DNA Methylation; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Receptors, Retinoic Acid; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Tropomyosins (TMs) are a family of microfilament binding proteins, which are suppressed in the transformed cells. We have investigated the mechanism of suppression of TMs, in particular that of tropomyosin-1 (TM1), in breast cancer cells. Inhibition of DNA methyl transferase with 5-aza-2'-deoxycytidine (AZA) alone did not induce TM1 expression. However, combined treatment of trichostatin A (TSA) and AZA resulted in readily detectable expression of TM1, but not that of other TM isoforms. Upregulation of TM1 expression paralleled with the reemergence of TM1 containing microfilaments, and in abolition of anchorage-independent growth. The synergistic action of AZA and TSA in reactivation of TM1 gene was also evident in ras-transformed fibroblasts. These data, for the first time, show that hypermethylation of TM1 gene and chromatin remodeling are the predominant mechanisms by which TM1 expression is downregulated in breast cancer cells.

Topics: Antibiotics, Antineoplastic; Azacitidine; Breast Neoplasms; Chromatin; Decitabine; DNA Methylation; Down-Regulation; Drosophila Proteins; Enzyme Inhibitors; Fibroblasts; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoblotting; Microscopy, Fluorescence; Promoter Regions, Genetic; Tropomyosin; Tumor Cells, Cultured

During tumorigenesis several cancer-related genes can be silenced by aberrant methylation. In many cases these silenced genes can be reactivated by exposure to the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AZA-CdR). Histone acetylation also plays a role in the control of expression of some genes. The aim of this study was to determine the antineoplastic activities of 5-AZA-CdR and trichostatin A (TSA), either administered alone or in combination. in MDA-MB-231 breast carcinoma cells. The effects of these drugs (alone and in combination) on the expression of the tumor suppressor gene, retinoic acid receptor (RAR beta) and of the estrogen receptor alpha gene (ER alpha), whose expression is lost in the cell line used in the study, were also investigated.. MDA-MB-231 cells were treated with 5-AZA-CdR and TSA and the antitumor activity of these drugs was determined by clonogenic assay. Total RNA was extracted from the treated cells and RT-PCR was used to determine the effect of the treatment on the expression of RAR beta and ER alpha. Methylation-sensitive PCR analysis was used to confirm that lack of expression of both genes was due to hypermethylation of their promoter regions. A single nucleotide primer extension assay was also used to quantify the reduction in DNA methylation following drug treatment.. Both 5-AZA-CdR and TSA alone showed significant antineoplastic activity. The combination of the two drugs was synergistic with respect to MDA-MB-231 cell kill. 5-AZA-CdR alone weakly activated the expression of both RAR beta and ER alpha. TSA alone only activated RAR beta, but not ER alpha. The combination of these agents appeared to produce a greater activation of both genes.. The interesting interaction between 5-AZA-CdR and TSA in both cell kill and cancer-related gene reactivation provides a rationale for the use of inhibitors of DNA methylation and histone deacetylation in combination for the chemotherapy of breast cancer.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Breast Neoplasms; Decitabine; Enzyme Inhibitors; Estrogen Receptor alpha; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Receptors, Estrogen; Receptors, Retinoic Acid; Tumor Cells, Cultured

Specific cell growth stimulators and inhibitors regulate IGF binding protein-3 (IGFBP-3), where in turn IGFBP-3 mediates their biological effects. The molecular mechanism(s) by which these factors regulate IGFBP-3 are unknown. Sodium butyrate, a histone deacetylase inhibitor causing growth arrest and differentiation, increases IGFBP-3 expression. We investigated the molecular mechanism of this induction using an IGFBP-3 promoter reporter system in MCF-7 and Hs578T breast cancer cells. IGFBP-3 promoter activity was induced up to 40-fold following a 24-h treatment with sodium butyrate and 46-fold in cells treated with trichostatin A, a pure histone deacetylase inhibitor. Deletion analysis of the IGFBP-3 promoter identified key sodium butyrate-responsive element(s) to a 45-bp region containing consensus binding sites for Sp1 and activating protein-2. Sp1 binding to the Sp1 site and Sp3 to the activating protein-2/GA-box played a functional role in sodium butyrate's activation of the IGFBP-3 promoter, however, with no change in binding direct sodium butyrate regulation was attributed to cofactors. The histone acetyltransferase p300 and histone deacetylase-1 were identified in multiprotein complexes containing DNA bound Sp1 and Sp3, with p300 accumulating following sodium butyrate treatment. Taken together, these data suggest that sodium butyrate increases IGFBP-3 expression by activating the IGFBP-3 promoter via an Sp1/Sp3 multiprotein complex, a mechanism that may be important for other key regulators of IGFBP-3.

Topics: Base Sequence; Breast Neoplasms; Butyrates; Cell Line; DNA-Binding Proteins; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Insulin-Like Growth Factor Binding Protein 3; Molecular Sequence Data; Promoter Regions, Genetic; Response Elements; Sp1 Transcription Factor; Sp3 Transcription Factor; Transcription Factors

Formation of transcriptional repression complexes such as DNA methyltransferase (DNMT) 1/histone deacetylase (HDAC) or methyl-CpG binding protein/HDAC is emerging as an important mechanism in silencing a variety of methylated tissue-specific and imprinted genes. Our previous studies showed that treatment of estrogen receptor (ER)-alpha-negative human breast cancer cells with the DNMT inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) led to ER mRNA and protein re-expression. Also, the HDAC inhibitor trichostatin A (TSA) could induce ER transcript about 5-fold. Here we show that 5-aza-dC alone induced ER transcript about 30-40-fold, and the addition of TSA elevated ER mRNA expression about 10-fold more in the human ER-negative breast cancer cell lines MDA-MB-231 and MDA-MB-435. Overall, the combination of 5-aza-dC and TSA induced a 300-400-fold increase in ER transcript. Restoration of estrogen responsiveness was demonstrated by the ability of the induced ER protein to elicit estrogen response element-regulated reporter activity from an exogenous plasmid as well as induce expression of the ER target gene, progesterone receptor. The synergistic activation of ER occurs concomitantly with markedly reduced soluble DNMT1 expression and activity, partial demethylation of the ER CpG island, and increased acetylation of histones H(3) and H(4). These data suggest that the activities of both DNMT1 and HDAC are key regulators of methylation-mediated ER gene silencing.

Topics: Acetylation; Azacitidine; Breast Neoplasms; CpG Islands; Decitabine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Drug Synergism; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Fulvestrant; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Receptors, Estrogen; RNA, Messenger; Tumor Cells, Cultured

There is a strong correlation between the acetylation status of nucleosomal histones and transcriptional activity. Here we show that the histone deacetylase inhibitor trichostatin A (TSA) activates reporter gene constructs driven by the human platelet-derived growth factor B (PDGF-B) gene promoter. This activation showed an inverse correlation with the cell type-specific transcriptional activities of the promoter. The TSA response was minimal in three tumor cell lines that exhibit high-level promoter activity. In JEG-3 choriocarcinoma cells, however, where the basal promoter activity is considerably lower, there was a strong response to TSA. This was in contrast to constructs that included a PDGF-B enhancer, which were refractory to TSA effects, indicating a possible function of the enhancer in modulating acetylation status. Analysis of PDGF-B promoter mutants with respect to TSA induction revealed no specific TSA-responsive element, but suggested that association of nonacetylated histones to the PDGF-B promoter may be a default process in the absence of enhancer activation. TSA treatment of JEG-3 cells, either alone or in combination with the demethylating agent 5-azacytidine, failed to activate the silenced endogenous PDGF-B transcript, however, which appears to be repressed by additional mechanisms.

Topics: Adenocarcinoma; Breast Neoplasms; Carcinoma, Hepatocellular; Choriocarcinoma; Chromosomes; DNA Methylation; Enhancer Elements, Genetic; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Introns; Liver Neoplasms; Mutagenesis; Promoter Regions, Genetic; Proto-Oncogene Proteins c-sis; Rhabdomyosarcoma; Transcription, Genetic; Tumor Cells, Cultured

Deregulation of the HER2 oncogene occurs in 30% of human breast cancers and correlates with poor prognosis and increased propensity for metastasis. Since the molecular basis of HER2 overexpression in human cancers is not known, we sought to determine whether chromatin remodeling pathways are involved in the regulation of HER2 expression. We report that compared with breast cancer cells expressing a low level of HER2, HER2-overexpressing breast cancer cells contained significantly higher levels of acetylated and phosphorylated histone H3, and acetylated histone H4 associated with the HER2 promoter. Decreased recruitment of histone deacetylases in the promoter is also noted in the HER2-overexpressing cell. The association of acetylated histone H4 with HER2 gene chromatin and HER2 expression in breast cancer cells was upregulated by an inhibitor of histone deacetylases. Treatment with histone deacetylase inhibitor also reduced the association of histone deacetylase-1 and -2 with the HER2 promoter. In addition, the tumor promoters 12-O-tetradecanoylphorbol-13-acetate and okadaic acid stimulated the association of phosphorylated histone H3 on serine 10 with the HER2 promoter and also stimulated HER2 expression. These findings identify histone acetylation and histone phosphorylation as novel regulatory modifications that target HER2 gene chromatin, and suggest that elevated levels of these chromatin-relaxing components in the vicinity of the HER2 gene promoter may constitute an important non-genomic mechanism of HER2 overexpression in human breast cancer.

Topics: Acetylation; Base Sequence; Breast Neoplasms; Chromatin; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-2; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Okadaic Acid; Phosphorylation; Promoter Regions, Genetic; Serine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The aromatic hydrocarbon receptor (AhR) is a ligand-dependent basic helix-loop-helix-PAS-containing transcription factor which is activated by chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Constitutive expression of the AhR gene occurs in a tissue- and developmentally specific manner and appears to be altered by chemicals which affect histone deacetylase (HDAC) activity in cells in culture. Here we have directly characterized the effects of two HDAC inhibitors, n-butyrate and trichostatin A, on the promoter activity of the murine AhR gene. HDAC inhibitors increased the constitutive activity of the AhR gene promoter in a luciferase reporter construct by five- to sevenfold in a dose- and time-dependent manner in several cell lines and was correlated with an increase in endogenous AhR activity in an AhR-deficient cell line. Deletion analysis of the upstream region of the AhR gene localized the HDAC inhibitor effect to a 167-bp region encompassing -77 to +90 of the AhR gene promoter. Cotransfection of an AhR promoter-luciferase reporter plasmid with a vector expressing the E1A(12s) oncoprotein, a negative regulator of p300, a protein with histone acetylase activity, decreased AhR promoter activity fivefold. Overall, our results support a role for histone acetylation in the transcriptional activity of the AhR gene promoter.

Topics: Animals; Breast Neoplasms; Butyrates; Carcinoma, Hepatocellular; COS Cells; Enzyme Inhibitors; Female; Gene Expression Regulation; Genes, Reporter; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kinetics; Liver Neoplasms; Luciferases; Mice; Okadaic Acid; Promoter Regions, Genetic; Receptors, Aryl Hydrocarbon; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Transfection; Tumor Cells, Cultured

Retinoic acid (RA)-resistance in breast cancer cells has been associated with irreversible loss of retinoic acid receptor beta, RARbeta, gene expression. Search of the causes affecting RARbeta gene activity has been oriented at identifying possible differences either at the level of one of the RARbeta promoters, RARbeta2, or at regulatory factors. We hypothesized that loss of RARbeta2 activity occurs as a result of multiple factors, including epigenetic modifications, which can pattern RARbeta2 chromatin state. Using methylation-specific PCR, we found hypermethylation at RARbeta2 in a significant proportion of both breast cancer cell lines and primary breast tumors. Treatment of cells with a methylated RARbeta2 promoter, by means of the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR), led to demethylation within RARbeta2 and expression of RARbeta indicating that DNA methylation is at least one factor, contributing to RARbeta inactivity. However, identically methylated promoters can differentially respond to RA, suggesting that RARbeta2 activity may be associated to different repressive chromatin states. This supposition is supported by the finding that the more stable repressive RARbeta2 state in the RA-resistant MDA-MB-231 cell line can be alleviated by the HDAC inhibitor, trichostatin A (TSA), with restoration of RA-induced RARbeta transcription. Thus, chromatin-remodeling drugs might provide a strategy to restore RARbeta activity, and help to overcome the hurdle of RA-resistance in breast cancer.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Breast; Breast Neoplasms; Cell Line; Chromatin; CpG Islands; Decitabine; DNA Methylation; Enzyme Inhibitors; Epithelial Cells; Female; Gene Expression Regulation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Receptors, Estrogen; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

The anticancer effects of retinoids are mainly mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are encoded by three distinct genes (alpha, beta, and gamma). Recent studies have demonstrated that RARbeta plays a critical role in mediating anticancer effects of retinoids. However, how RARbeta exerts its potent anticancer effects remains largely unknown. In this study, we investigated anti-Activator Protein-1 (AP-1) activity of RARbeta. In a transient transfection assay, all three RAR subtypes, RARalpha, RARbeta, and RARgamma, could effectively inhibit phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity and the activity of oncogenes c-Jun and c-Fos on AP-1 containing reporter genes in the presence of retinoic acid (RA). However, RARbeta showed a strong RA-independent inhibition of AP-1 activity, whereas inhibition of AP-1 activity by RARalpha and RARgamma was RA dependent. By using several hybrid receptors that contain either the COOH-terminal portion or the NH2-terminal portion of RARbeta, we demonstrated that the NH2-terminal portion of RARbeta, the A/B domain, was mainly responsible for the RA-independent inhibition of AP-1 activity. This activity was not attributable to constitutive AF-1 activity of RARbeta, because it did not activate several RA response element-containing reporter genes. In addition, inhibition of histone deacetylase activity by trichostatin A did not overcome the inhibitory effect of RARbeta. In cancer cells, stable transfection of RARbeta exhibited strong inhibition of AP-1 activity, even in the absence of RA. Moreover, expression of endogenous AP-1-responsive gene collagenase I was strongly repressed in cancer cells stably transfected with RARbeta. In studying the antitransforming activity of RARbeta, we observed that the growth of breast cancer MDA-MB231 cells in soft agar was significantly repressed in a RA-independent manner when cells were stably transfected with RARbeta but not RARalpha. Together, our results demonstrate that RARbeta may exert its potent anticancer effect in part through its unique anti-AP-1 activity.

Topics: Breast Neoplasms; Carcinogens; Cell Division; Collagenases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genes, Reporter; HeLa Cells; Histone Deacetylases; Humans; Hydroxamic Acids; Mutagenesis; Plasmids; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Reverse Transcriptase Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transfection; Tretinoin; Tumor Cells, Cultured

Quinidine inhibits proliferation and promotes cellular differentiation in human breast tumor epithelial cells. Previously we showed quinidine arrested MCF-7 cells in G(1) phase of the cell cycle and led to a G(1) to G(0) transition followed by apoptotic cell death. The present experiments demonstrated that MCF-7, MCF-7ras, T47D, MDA-MB-231, and MDA-MB-435 cells transiently differentiate before undergoing apoptosis in response to quinidine. The cells accumulated lipid droplets, and the cytokeratin 18 cytoskeleton was reorganized. Hyperacetylated histone H4 appeared within 2 h of the addition of quinidine to the medium, and levels were maximal by 24 h. Quinidine-treated MCF-7 cells showed elevated p21(WAF1), hypophosphorylation and suppression of retinoblastoma protein, and down-regulation of cyclin D1, similar to the cell cycle response observed with cells induced to differentiate by histone deacetylase inhibitors, trichostatin A, and trapoxin. Quinidine did not show evidence for direct inhibition of histone deacetylase enzymatic activity in vitro. HDAC1 was undetectable in MCF-7 cells 30 min after addition of quinidine to the growth medium. The proteasome inhibitors MG-132 and lactacystin completely protected HDAC1 from the action of quinidine. We conclude that quinidine is a breast tumor cell differentiating agent that causes the loss of HDAC1 via a proteasomal sensitive mechanism.

Topics: Acetylation; Acetylcysteine; Animals; Anti-Bacterial Agents; Breast Neoplasms; Cell Cycle; Cell Differentiation; Cell Division; Chickens; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoskeleton; Down-Regulation; Enzyme Inhibitors; Female; G1 Phase; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Immunoblotting; Keratins; Leupeptins; Multienzyme Complexes; Peptides; Phosphorylation; Proteasome Endopeptidase Complex; Quinidine; Retinoblastoma Protein; Time Factors; Tumor Cells, Cultured

Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994). In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase 1-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1-2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.

Topics: Animals; Base Sequence; Binding Sites; Breast Neoplasms; Cell Division; Chromatin; Deoxyribonuclease I; DNA Methylation; DNA, Neoplasm; Drug Resistance, Neoplasm; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogens; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Luciferases; Molecular Sequence Data; Mutation; Neoplasms, Hormone-Dependent; Plasmids; Promoter Regions, Genetic; Protein Biosynthesis; Proteins; Receptors, Estradiol; Receptors, Progesterone; Tamoxifen; Thymidine Kinase; Time Factors; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins; Vitellogenins; Xenopus

Recent findings have established a connection between DNA methylation and transcriptionally inactive chromatin characterized by deacetylated histones. Because the absence of estrogen receptor alpha (ERalpha) gene expression has been associated with aberrant methylation of its CpG island in a significant fraction of breast cancers, we tested whether histone deacetylase activity contributes to the transcriptional inactivation of the methylated ER gene in a panel of ER-negative human breast cancer cells. Treatment of these cells with trichostatin A, a specific histone deacetylase inhibitor, led to dose- and time-dependent re-expression of ER mRNA as detected by reverse transcription-PCR without alteration in ERalpha CpG island methylation. Trichostatin A-induced ER re-expression was associated with increased sensitivity to DNase I at the ER locus in MDA-MB-231 cells. These data implicate inactive chromatin mediated by histone deacetylation as a critical component of ER gene silencing in human breast cancer cells. Therefore, histone deacetylation may be a potential target for therapeutic intervention in the treatment of a subset of ER-negative breast cancers.

Topics: Base Sequence; Breast Neoplasms; Chromatin; CpG Islands; Deoxyribonuclease I; DNA Methylation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrogen Receptor alpha; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Molecular Sequence Data; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transcriptional Activation; Tumor Cells, Cultured

Topics: Biomarkers, Tumor; Breast Neoplasms; Cyclin D1; Cytoskeleton; Down-Regulation; Gelsolin; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Tumor Cells, Cultured

Inhibitors of histone deacetylase are attracting increasing interest due to their influence on transcription, differentiation, and apoptosis. We have investigated two synthetic inhibitors 3 and 4 of histone deacetylase and the natural product inhibitor trichostatin A for their ability to suppress the growth of MCF-7 breast cancer cells and here present complete and improved synthetic procedures. The compounds show a dose dependent inhibition of growth with activities in the low micromolar and nanomolar range. Trichostatin shows cytocidal effects at 100 nM and still has activity comparable to cisplatin (0.5 microM) at 10 nM. Whereas the synthetic inhibitor 3 has cytocidal activity at 10 microM compound 4 shows a maximum of 40% growth suppression at that concentration.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Division; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Tumor Cells, Cultured

Nuclear receptor corepressor (NCoR) mediates repression (silencing) of basal gene transcription by nuclear receptors for thyroid hormone and retinoic acid. The goal of this study was to create novel estrogen receptor (ER) mutants by fusing transferable repressor domains from the N-terminal region of NCoR to a functional ER fragment. Three chimeric NCoR-ER proteins were created and shown to lack transcriptional activity. These fusion proteins silenced basal transcription of the ERE2-tk-Luc reporter gene and inhibited the activity of co-transfected wild-type ER (wtER), indicating that they possess dominant negative activity. One of the fusion proteins (CDE-RD1), containing the ER DNA-binding and ligand-binding domains linked to the NCoR repressor domain (RD1), was selected for detailed examination. Its hormone affinity, intracellular localization, and level of expression in transfected cells were similar to wtER, and it bound to the estrogen response element (ERE) DNA in gel shift assays. Glutathione-S-transferase pull-down assays showed that CDE-RD1 retains the ability to bind to steroid receptor coactivator-1. Introduction of a DNA-binding domain mutation into the CDE-RD1 fusion protein eliminated silencing and dominant negative activity. Thus, the RD1 repressor domain prevents transcriptional activation despite the apparent ability of CDE-RD1 to bind DNA, ligand, and coactivators. Transcriptional silencing was incompletely reversed by trichostatin A, suggesting a histone deacetylase-independent mechanism for repression. CDE-RD1 inhibited ER-mediated transcription in T47D and MDA-MB-231 breast cancer cells and repressed the growth of T47D cells when delivered to the cells by a retroviral vector. These ER-NCoR fusion proteins provide a novel means for inhibiting ER-mediated cellular responses, and analogous strategies could be used to create dominant negative mutants of other transcription factors.

Topics: Binding Sites; Breast Neoplasms; Cell Division; Cell Line; DNA; Enzyme Inhibitors; Estradiol; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kidney; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Coactivator 1; Receptors, Estrogen; Recombinant Fusion Proteins; Repressor Proteins; Response Elements; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured