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Recent evidence indicates that tumor-initiating cells (TICs), also called cancer stem cells (CSCs), are responsible for tumor initiation and progression, therefore representing an important cell population that may be used as a target for the development of future anticancer therapies. In the present study, Cryptotanshinone (CT), a traditional Chinese herbal medicine, was demonstrated to regulate the behaviors of LNCaP prostate cells and prostate LNCaP TICs. The results demonstrate that treatment with CT alters cellular proliferation, cell cycle status, migration, viability, colony formation and notably, sphere formation and down-regulation of stemness genes (Nanog, OCT4, SOX2, β-catenin, CXCR4) in TICs. The present study demonstrates that CT targets the LNCaP CD44+CD24- population that is representative of prostate TICs and also affects total LNCaP cells as well via down-regulation of stemness genes. The strong effect with which CT has on prostate TICs suggests that CT may potentially function as a novel natural anticancer agent that specifically targets TICs.

Metastatic prostate cancer (PCa) is still an incurable disease. Long non-coding RNAs (lncRNAs) may be an overlooked source of cancer biomarkers and therapeutic targets. We therefore performed RNA sequencing on paired metastatic/non-metastatic PCa xenografts derived from clinical specimens. The most highly up-regulated transcript was LOC728606, a lncRNA now designated PCAT18. PCAT18 is specifically expressed in the prostate compared to 11 other normal tissues (p<0.05) and up-regulated in PCa compared to 15 other neoplasms (p<0.001). Cancer-specific up-regulation of PCAT18 was confirmed on an independent dataset of PCa and benign prostatic hyperplasia samples (p<0.001). PCAT18 was detectable in plasma samples and increased incrementally from healthy individuals to those with localized and metastatic PCa (p<0.01). We identified a PCAT18-associated expression signature (PES), which is highly PCa-specific and activated in metastatic vs. primary PCa samples (p<1E-4, odds ratio>2). The PES was significantly associated with androgen receptor (AR) signalling. Accordingly, AR activation dramatically up-regulated PCAT18 expression in vitro and in vivo. PCAT18 silencing significantly (p<0.001) inhibited PCa cell proliferation and triggered caspase 3/7 activation, with no effect on non-neoplastic cells. PCAT18 silencing also inhibited PCa cell migration (p<0.01) and invasion (p<0.01). These results position PCAT18 as a potential therapeutic target and biomarker for metastatic PCa.

Pancreatic cancer is the fourth leading cause of cancer-related mortality in the world. Pancreatic cancer can be localized, locally advanced, or metastatic. The median 1- and 5-year survival rates are 25% and 6%, respectively. Epigenetic modifications such as DNA methylation play a significant role during both normal human development and cancer progression. To investigate epigenetic regulation of genes in the tumor-initiating population of pancreatic cancer cells, which are also termed cancer stem cells (CSCs), we conducted epigenetic arrays in PANC1 and HPAC pancreatic cancer cell lines and compared the global DNA methylation status of CpG promoters in invasive cells, demonstrated to be CSCs, to their noninvasive counterparts, or non-CSCs. Our results suggested that the NF-κB pathway is one of the most activated pathways in pancreatic CSCs. In agreement with this, we determined that upon treatment with NF-κB pathway inhibitors, the stem cell-like properties of cells are significantly disrupted. Moreover, SOX9, demethylated in CSCs, is shown to play a crucial role in the invasion process. Additionally, we found a potential NF-κB binding site located in the SOX9 promoter and determined that the NF-κB subunit p65 positively regulates SOX9 expression by binding to its promoter directly. This interaction can be efficiently blocked by NF-κB inhibitors. Thus, our work establishes a link between the classic NF-κB signaling transduction pathway and the invasiveness of pancreatic CSCs, which may result in the identification of novel signals and molecules that function at an epigenetic level, and could potentially be targeted for pharmaceutical investigations and clinical trials., (Copyright © 2013 AlphaMed Press.)

Histone lysine demethylases (KDMs) have been recently discovered in mammals and have been nicknamed "erasers" for their ability to remove methyl groups from histone substrates. In cancer cells, KDMs can activate or repress gene transcription, behaving as oncogenes or tumor suppressors depending upon the cellular context. In order to investigate the potential role of KDMs in Breast Cancer (BC), we queried the Oncomine database and determined that the expression of KDMs correlates with BC prognosis. High expression of KDM3B and KDM5A is associated with a better prognosis (no recurrence after mastectomy p=0.005 and response to docetaxel p=0.005); conversely, KDM6A is overexpressed in BC patients with an unfavorable prognosis (mortality at 1 year, p=8.65E-7). Our findings suggest that KDMs could be potential targets for BC therapy. Further, altering the interactions between KDMs and Polycomb Group genes (PcG) may provide novel avenues for therapy that specifically targets these genes in BC., (Published by Elsevier Ireland Ltd.)

Early prostate cancer (PCa) is generally treatable and associated with good prognosis. After a variable time, PCa evolves into a highly metastatic and treatment-refractory disease: castration-resistant PCa (CRPC). Currently, few prognostic factors are available to predict the emergence of CRPC, and no curative option is available. Epigenetic gene regulation has been shown to trigger PCa metastasis and androgen-independence. Most epigenetic studies have focused on DNA and histone methyltransferases. While DNA methylation leads to gene silencing, histone methylation can trigger gene activation or inactivation, depending on the target amino acid residues and the extent of methylation (me1, me2, or me3). Interestingly, some histone modifiers are essential for PCa tumor-initiating cell (TIC) self-renewal. TICs are considered the seeds responsible for metastatic spreading and androgen-independence. Histone Lysine Demethylases (KDMs) are a novel class of epigenetic enzymes which can remove both repressive and activating histone marks. KDMs are currently grouped into 7 major classes, each one targeting a specific methylation site. Since their discovery, KDM expression has been found to be deregulated in several neoplasms. In PCa, KDMs may act as either tumor suppressors or oncogenes, depending on their gene regulatory function. For example, KDM1A and KDM4C are essential for PCa androgen-dependent proliferation, while PHF8 is involved in PCa migration and invasion. Interestingly, the possibility of pharmacologically targeting KDMs has been demonstrated. In the present paper, we summarize the emerging role of KDMs in regulating the metastatic potential and androgen-dependence of PCa. In addition, we speculate on the possible interaction between KDMs and other epigenetic effectors relevant for PCa TICs. Finally, we explore the role of KDMs as novel prognostic factors and therapeutic targets. We believe that studies on histone demethylation may add a novel perspective in our efforts to prevent and cure advanced PCa.

Background: Despite therapeutic innovations, metastatic colorectal cancer (mCRC) is still characterized by poor prognosis and few molecular markers predict the risk of progression. Polycomb group genes (PcGs) are epigenetic modifiers involved in tumor suppressor gene silencing. PcG member EZH2 mediates gene silencing through histone-H3 lysine-27 methylation. In colorectal cancer (CRC), EZH2 overexpression predicts shorter survival. Recently, four EZH2 single-nucleotide polymorphisms (SNPs) have been described. The present study was aimed at evaluating the correlation between EZH2 SNPs and outcome parameters in mCRC patients., Patients and Methods: DNA was extracted from blood samples of 110 mCRC patients treated with first-line 5-fluorouracil, folinic acid, irinotecan (FOLFIRI) and bevacizumab. Genotyping was carried out by real-time PCR. Genotype was used to predict objective response, progression-free survival (PFS) and overall survival (OS). EZH2 messenger RNA levels were evaluated on lymphocytes of a parallel cohort of 50 CRC patients., Results: One allelic variant (rs3757441 C/C versus C/T or T/T) was significantly associated with shorter PFS and OS (P < 0.01 and P < 0.05, respectively). At multivariate analysis, the same variant resulted an independent predictor of PFS and OS (P < 0.05). The C/C variant was associated with significantly higher EZH2 expression (P < 0.05)., Conclusion: An EZH2 SNP may be useful to predict clinical outcome in mCRC patients.

TICs are characterized by their ability to self-renew, differentiate and initiate tumor formation. miRNAs are small noncoding RNAs that bind to mRNAs resulting in regulation of gene expression and biological functions. The role of miRNAs and TICs in cancer progression led us to hypothesize that miRNAs may regulate genes involved in TIC maintenance. Using whole genome miRNA and mRNA expression profiling of TICs from primary prostate cancer cells, we identified a set of up-regulated miRNAs and a set of genes down-regulated in PSs. Inhibition of these miRNAs results in a decrease of prostatosphere formation and an increase in target gene expression. This study uses genome-wide miRNA profiling to analyze expression in TICs. We connect aberrant miRNA expression and deregulated gene expression in TICs. These findings can contribute to a better understanding of the molecular mechanisms governing TIC development/maintenance and the role that miRNAs have in the fundamental biology of TICs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Polycomb repressive complex 2 (PRC2) mediates gene silencing through histone H3K27 methylation. PRC2 components are over-expressed in metastatic prostate cancer (PC), and are required for cancer stem cell (CSC) self-renewal. 3-Dezaneplanocin-A (DZNeP) is an inhibitor of PRC2 with broad anticancer activity., Method: we investigated the effects of DZNeP on cell proliferation, tumorigenicity and invasive potential of PC cell lines (LNCaP and DU145)., Results: Exploring GEO and Oncomine databases, we found that specific PRC2 genes (EED, EZH2, SUZ12) predict poor prognosis in PC. Non-toxic DZNeP concentrations completely eradicated LNCaP and DU145 prostatosphere formation, and significantly reduced the expression of CSC markers. At comparable doses, other epigenetic drugs were not able to eradicate CSCs. DZNeP was also able to reduce PC cell invasion. Cells pre-treated with DZNeP were significantly less tumorigenic (LNCaP) and formed smaller tumors (DU145) in immunocompromised mice., Conclusion: DZNeP is effective both in vitro and in vivo against PC cells. DZNeP antitumor activity is in part mediated by inhibition of CSC tumorigenic potential.

Background: Recently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs) that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation., Results: Differentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1., Conclusions: Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells. These aggressive sub-populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis.

Polycomb group (PcG) proteins are crucial for neural cancer stem cell (NCSC) self-renewal. However, the relative expression levels of PcG genes in different subtypes of brain tumors, their prognostic role and their effects on cellular pathways have not been investigated. For this purpose, we queried the Oncomine database and found that 4 PcG genes (EZH2, RBBP7, SUZ12, YY1) are specifically expressed in brain tumors. EZH2 expression increases with tumor grade in adult and pediatric brain tumors, and is a poor prognostic factor. In glioblastoma, EZH2 inhibits differentiation, and activates cancer-, cell cycle- and cellular movement-related genes. In keeping with previously published data, our results suggest that EZH2 is both a prognostic factor and a promising therapy target in brain tumors.

Background: The cancer stem cell (CSC) hypothesis proposes that a population of tumor cells bearing stem cell properties is responsible for the origin and maintenance of tumors. Normal and cancer stem cells possess the ability to grow in vitro as self-renewing spheres, but the molecular basis of this phenotype remains largely unknown. We intended to establish a comprehensive culture system to grow prostatospheres (PSs) from both cancer cell lines and patient tumors. We then used gene expression microarrays to gain insight on the molecular pathways that sustain the PS tumor initiating cell (TIC) phenotype., Results: Traditional stem cell medium (SCM) supplemented with KnockoutSR (KO) allows the propagation of monoclonal PSs from cell lines and primary cells. PSs display gene expression and tumorigenicity hallmarks of TICs. Gene expression analysis defined a gene signature composed of 66 genes that characterize LNCaP and patient PSs. This set includes novel prostate TIC growth factors (NRP1, GDF1, JAG1), proteins implicated in cell adhesion and cytoskeletal maintenance, transcriptional regulators (MYCBP, MYBL1, ID1, ID3, FOS, ELF3, ELF4, KLF2, KLF5) and factors involved in protein biosynthesis and metabolism. Meta-analysis in Oncomine reveals that some of these genes correlate with prostate cancer status and/or progression. Reporter genes and inhibitors indicate that the Notch pathway contributes to prostatosphere growth., Conclusions: We have developed a model for the culture of PSs, and provide a genomic profile that support CSCs identity. This signature identifies novel markers and pathways that are predicted to correlate with prostate cancer evolution.

There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self-renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin alpha V beta 3-dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of beta-catenin nuclear localization. Blocking integrin alpha V beta 3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways.

Prostate cancer continues to represent a burgeoning medical problem in the United States. Recent studies suggest that gossypol, a bioactive phytochemical produced by cotton plants, is a promising agent against prostate cancer. The current studies were undertaken to examine the chemotherapeutic efficacy of gossypol on human prostate cancer cell lines and prostate tumor-initiating cells. Gossypol reduced the viability of three prostate cancer cell lines (LAPC4, PC3, and DU145) with an IC(50) between 3 and 5 micromol/L. Additionally, gossypol was effective at inhibiting prostate tumor-initiating cell-driven tumor growth in a nonobese diabetic/severe combined immunodeficient xenograft model. Our integrated molecular profiling approach encompassing proteomics, activated transcription factors, and genomics suggests that the decrease in viability was associated with increased DNA damage and the induction of apoptosis. Exposure of DU145 cells to gossypol (1-10 micromol/L) resulted in the activation of 13 proteins and 7 transcription factors, and the expression of 17 genes involved in the mitochondrial pathway of apoptosis. These studies show for the first time that gossypol treatment induces DNA damage and activates p53. Collectively, these data support the use of gossypol as a novel agent for prostate cancer.

Cancer stem cells (CSCs) are thought to sustain cancer progression, metastasis and recurrence after therapy. There is in vitro and in vivo evidence supporting the idea that CSCs are highly chemoresistant. Epigenetic gene regulation is crucial for both stem cell biology and chemoresistance. In this review, we summarize current data on epigenetic mechanisms of chemoresistance in cancer stem cells. We propose a model integrating classical CSC pathways (Wnt, Hedgehog and Notch), epigenetic effectors (Polycomb) and drug resistance genes (ABCG2, CD44). Moreover, we analyze the potential of epigenetic drugs to reverse CSC chemoresistance. In the future, CSC epigenomic profiling could help to dissect specific chemoresistance pathways, and have a significant clinical impact for patient stratification and rational design of therapeutic regimens.

Aberrantly activated signal transducer and activator of transcription 3 (Stat3) is implicated in the development of various human cancers. Y705 phosphorylation is conventionally thought to be required for Stat3 signal-dependent activation and seems to play an essential role in some malignancies. Recently, it was shown that Stat3 is activated through novel and noncanonical mechanisms, including phosphorylation at S727. Here, we investigate S727 phosphorylation of Stat3 and its subsequent effects in prostate cancer development, independent of Y705 phosphorylation, using mutated Stat3 in the human prostate cancer cell line LNCaP. We show mutation of S727 to the phosphomimetic residue Glu, and inactivation of Y705 (Y705F/S727E) resulted in a remarkable growth advantage in low-serum, enhanced anchorage-independent growth in soft agar, and increased tumorigenicity in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, possibly by direct activation of downstream proto-oncogenes c-myc, mcl-1, and survivin. Y705F/S727E mutant cells were more invasive than Y705F/S727A (inactivation of Y705 and S727) mutant cells, and more Y705F/S727E mutant Stat3 was localized in the nuclei relative to Y705F/S727A mutant Stat3 at the steady state. Furthermore, the Y705F/S727E but not the Y705F/S727A mutant induced anchorage-independent growth of noncancerous prostate epithelial cells (RWPE-1). We further show that Stat3 is phosphorylated at S727 in 65% of malignant prostate tissues (n = 20) relative to 25% of normal prostate tissues (n = 4). Moreover, there is a positive correlation between phosphoS727-Stat3 expression and Gleason score in these prostate cancer tissues (P = 0.05). Our data suggest for the first time that S727 phosphorylation is sufficient to activate Stat3, thereby driving prostate tumorigenesis independent of Y705 phosphorylation.

Purpose: Advanced prostate cancer is first treated with androgen deprivation therapy. However, tumors become resistant to and grow despite castrate levels of testosterone. Growth and proliferation of CRPC is mediated by gain-of-function changes in the AR and AR reactivation. Expression of manganese superoxide dismutase (SOD2), which regulates cellular ROS, is markedly down-regulated in CRPC when compared with hormone-responsive tumors., Experimental Design: Here, we knocked down SOD2 expression in AR-expressing LNCaP prostate cancer cells and determined gene expression changes, transcription factor binding, and AR transcription activity in SOD2 knockdown cells., Results: SOD2 knockdown results in an increase in ROS. Gene expression changes induced by SOD2 knockdown results in the up-regulation of genes that are also androgen responsive and 46% of genes up-regulated 2-fold by the androgen ligand R1881 are also up-regulated to the same extent with SOD2 knockdown. The induction of many of these genes with SOD2 knockdown, such as VEGFA and FKBP5, is reversible with the antioxidant N-acetylcysteine, suggesting that this mechanism is directly linked to ROS. Furthermore, an array for transcription factor DNA-binding activity shows that SOD2 knockdown induces DNA binding by several transcription factors, including AR. SOD2 knockdown-induced AR activation was confirmed by electrophoretic mobility shift assay and luciferase activity, and both were readily reversible with N-acetylcysteine., Conclusions: These findings show that down-regulation of SOD2 induces AR activity in a ROS-dependent manner, and suggest that there may be a role for antioxidant therapy in CRPC.

Cancer, second only to heart disease, is the leading cause of death in the US. Although progress has been made in the early detection of cancer and in improvements of cancer therapies, the ability to provide long-term survival has been limited. Increasing evidence suggests that a minute, biologically unique population of cancer stem cells (SCs) exists in most neoplasms and may be responsible for tumor initiation, progression, metastasis, and relapse. Characterization of cancer SCs has led to the identification of key cellular activities that may make cancer SCs vulnerable to therapeutic interventions that target drug-effluxing capabilities, stem cell pathways, anti-apoptotic mechanisms, and induction of differentiation. Phytochemicals, compounds made from fruits, vegetables, and grains, possess anti-cancer properties and represent a promising therapeutic approach for the prevention and treatment of many cancers. This review summarizes the evidence for the cancer SC hypothesis and discusses the potential mechanisms by which phytochemicals might target cancer SCs.

Breast cancer is one of the most common malignancies in women. Despite advances in treatment of endocrine-dependent tumors, the complete molecular basis of transformation is still unknown. What is clear is that a variety of genetic lesions and epigenetic modifications are present in the neoplasm. Disregulation of several signaling pathways is known to be associated with breast cancer development, among them is the wingless and integration site growth factor (Wnt) pathway. While genetic mutations of certain components of this pathway, such as APC, are significant contributing factors for colorectal cancers, they are typically not the predominate mechanism associated with breast cancer. Instead, it appears that DNA hypermethylation leads to aberrant regulation of the Wnt pathway in breast cancer, and as such, this review focuses on the epigenetic regulation of Wnt pathway components in breast cancer.

Recent evidence supports the hypothesis that cancer stem cells are responsible for tumour initiation and formation. Using flow cytometry, we isolated a population of CD44+CD24(-) prostate cells that display stem cell characteristics as well as gene expression patterns that predict overall survival in prostate cancer patients. CD44+CD24(-) cells form colonies in soft agar and form tumours in NOD/SCID mice when as few as 100 cells are injected. Furthermore, CD44+CD24(-) cells express genes known to be important in stem cell maintenance, such as BMI-1 and Oct-3/4. Moreover, we can maintain CD44+CD24(-) prostate stem-like cells as nonadherent spheres in serum-replacement media without substantially shifting gene expression. Addition of serum results in adherence to plastic and shifts gene expression patterns to resemble the differentiated parental cells. Thus, we propose that CD44+CD24(-) prostate cells are stem-like cells responsible for tumour initiation and we provide a genomic definition of these cells and the differentiated cells they give rise to. Furthermore, gene expression patterns of CD44+CD24(-) cells have a genomic signature that is predictive of poor patient prognosis. Therefore, CD44+CD24(-) LNCaP prostate cells offer an attractive model system to both explore the biology important to the maintenance and differentiation of prostate cancer stem cells as well as to develop the therapeutics, as the gene expression pattern in these cells is consistent with poor survival in prostate cancer patients.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor and a member of the nuclear receptor superfamily. PPARgamma and its ligands appear to serve diverse biological functions. In addition to the well-studied effects of PPARgamma on metabolism and cellular differentiation, abundant evidence suggests that PPARgamma is an important regulator of the immune system and cancers. Since cytokines are not only key modulators of inflammation with pro- and anti-inflammatory functions but they also can either stimulate or inhibit tumor growth and progression, this review summarizes the role for PPARgamma in the regulation of cytokine production and cytokine-mediated signal transduction pathways in immune cells and cancer.

Binding of multiple myeloma (MM) cells to bone marrow stromal cells (BMSCs) triggers expression of adhesive molecules and secretion of interleukin-6 (IL-6), promoting MM cell growth, survival, drug resistance, and migration, which highlights the possibility of developing and validating novel anti-MM therapeutic strategies targeting MM cells-host BMSC interactions and their sequelae. Recently, we have found that expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands can potently inhibit IL-6-regulated MM cell growth. Here we demonstrate that PPARgamma agonists 15-d-PGJ2 and troglitazone significantly suppress cell-cell adhesive events, including expression of adhesion molecules and IL-6 secretion from BMSCs triggered by adhesion of MM cells, as well as overcome drug resistance by a PPARgamma-dependent mechanism. The synthetic and natural PPARgamma agonists have diverging and overlapping mechanisms blocking transactivation of transcription factors NF-kappaB and 5'-CCAAT/enhancer-binding protein beta (C/EBPbeta). Both 15-d-PGJ2 and troglitazone blocked C/EBPbeta transcriptional activity by forming PPARgamma complexes with C/EBPbeta. 15-d-PGJ2 and troglitazone also blocked NF-kappaB activation by recruiting the coactivator PGC-1 from p65/p50 complexes. In addition, 15-d-PGJ2 had a non-PPARgamma-dependent effect by inactivation of phosphorylation of IKK and IkappaB. These studies provide the framework for PPARgamma-based pharmacological strategies targeting adhesive interactions of MM cells with the bone marrow microenvironment.

The epigenetic programming of genomic DNA is accomplished, in part, by several DNA cytosine-5-methyltransferases that act by covalently modifying cytosines with the addition of a methyl group. This covalent modification is maintained by the DNA cytosine-5-methyltransferase-1 enzyme (DNMT1), which is capable of acting in concert with other similar enzymes to silence important tumor suppressor genes. IL-6 is a multifunctional mediator of inflammation, acting through several major signaling cascades, including the phosphatidylinositol-3-kinase pathway (PI-3-K), which activates protein kinase B (AKT/PKB) downstream. Here, we show that the subcellular localization of DNMT1 can be altered by the addition of IL-6, increasing the rate of nuclear translocation of the enzyme from the cytosolic compartment. The mechanism of nuclear translocation of DNMT1 is greatly enhanced by phosphorylation of the DNMT1 nuclear localization signal (NLS) by PKB/AKT kinase. Mutagenic alteration of the two AKT target amino acids within the NLS results in a major loss of DNMT1 nuclear translocation, while the creation of a "phospho-mimic" amino acid (mutation to acidic residues) restores this compartmentation ability. These observations suggest an interesting hypothesis regarding how mediators of chronic inflammation may disturb the delicate balance of cellular compartmentalization of important proteins, and reveals a potential mechanism for the induction or enhancement of tumor growth via alteration of the components involved in the epigenetic programming of a cell.

The transforming growth factor-beta (TGF-beta) pathway plays dual roles in cancer, inhibiting epithelial cell growth under normal physiologic conditions, but promoting invasion and metastasis once growth inhibitory responses are lost. Two recent papers show that TGF-beta receptor III is the most common TGF-beta pathway component downregulated in prostate cancer. Here, we discuss the implications of these findings and what it may mean about the biology of this disease.

Manganese superoxide dismutase (SOD2) is an enzyme that catalyses the dismutation of superoxide in the mitochondria, leading to reduced levels of reactive oxygen species. Reduced expression levels of SOD2 have been shown to result in increased DNA damage and sod2 heterozygous mice have increased incidences of cancer. It has also been shown that SOD2 expression is lost in pancreatic cell lines, with reintroduction of SOD2 resulting in decreased rate of proliferation. The mechanism of decreased SOD2 expression in pancreatic carcinoma has not been previously determined. We demonstrate, through sodium bisulphite sequencing, that the sod2 locus is methylated in some pancreatic cell lines leading to a corresponding decrease in SOD2 expression. Methylation can be reversed by treatment with zebularine, a methyltransferase inhibitor, resulting in restored SOD2 expression. Furthermore, we demonstrate that sensitivity of pancreatic carcinoma cell lines to 2-methoxyestradiol correlates with SOD2 expression and SOD2 modulation can alter the sensitivity of these cells. Using both genomics and proteomics, we also identify molecular consequences of SOD2 expression in MIA-PaCa2 cells, including dephosphorylation of VEGFR2 and the identification of both SOD2-regulated genes and transcription factors with altered binding activity in response to SOD2 expression.

Castrate-resistant prostate cancer (CRPC) continues to be dependent on the androgen receptor (AR) for disease progression. We have synthesized and evaluated a novel compound that is a conjugate of colchicine and an AR antagonist (cyanonilutamide) designed to inhibit AR function in CRPC. A problem in multifunctional AR-binding compounds is steric hindrance of binding to the embedded hydrophobic AR ligand-binding pocket. Despite the bulky side chain projecting off of the AR-binding moiety, this novel conjugate of colchicine and cyanonilutamide binds to AR with a K(i) of 449 nmol/L. Structural modeling of this compound in the AR ligand-binding domain using a combination of rational docking, molecular dynamics, and steered molecular dynamics simulations reveals a basis for how this compound, which has a rigid alkyne linker, is able to bind to AR. Surprisingly, we found that this compound also binds to tubulin and inhibits tubulin function to a greater degree than colchicine itself. The tubulin-inhibiting activity of this compound increases cytoplasmic AR levels in prostate cancer cells. Finally, we found that this compound has greater toxicity against androgen-independent prostate cancer cells than the combination of colchicine and nilutamide. Together, these data point to several ways of inhibiting AR function in CRPC.

Reactive oxygen species are known to be involved in several cellular processes, including cell signaling. SOD2 is a key enzyme in the conversion of reactive oxygen species and has been implicated in a host of disease states, including cancer. Using an integrated, whole-cell approach encompassing epigenetics, genomics, and proteomics, we have defined the role of SOD2 in multiple myeloma. We show that the SOD2 promoter is methylated in several cell lines and there is a correlative decrease in expression. Furthermore, myeloma patient samples have decreased SOD2 expression compared with healthy donors. Overexpression of SOD2 results in decreased proliferation and altered sensitivity to 2-methoxyestradiol-induced DNA damage and apoptosis. Genomic profiling revealed regulation of 65 genes, including genes involved in tumorigenesis, and proteomic analysis identified activation of the JAK/STAT pathway. Analysis of nearly 400 activated transcription factors identified 31 transcription factors with altered DNA binding activity, including XBP1, NFAT, forkhead, and GAS binding sites. Integration of data from our gestalt molecular analysis has defined a role for SOD2 in cellular proliferation, JAK/STAT signaling, and regulation of several transcription factors.

The cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) inhibits proliferation of cancer cells, including breast cancers, by peroxisome proliferator-activated receptor-gamma (PPARgamma)-dependent and PPARgamma-independent mechanisms. However, little is known about its effect on the transcriptional activity of estrogen receptor-alpha (ERalpha) that plays vital roles in the growth of breast cancers. Here, we show that 15d-PGJ(2) inhibits both 17beta-estradiol (E(2))-dependent and E(2)-independent ERalpha transcriptional activity by PPARgamma-independent mechanism. In addition, 15d-PGJ(2) directly modifies ERalpha protein via its reactive cyclopentenone moiety, evidenced by incorporation of biotinylated 15d-PGJ(2) into ERalpha, both in vitro and in vivo. Nanoflow reverse-phase liquid chromatography tandem mass spectrometry analysis identifies two cysteines (Cys(227) and Cys(240)) within the COOH-terminal zinc finger of ERalpha DNA-binding domain (DBD) as targets for covalent modification by 15d-PGJ(2). Gel mobility shift and chromatin immunoprecipitation assays show that 15d-PGJ(2) inhibits DNA binding of ERalpha and subsequent repression of ERalpha target gene expression, such as pS2 and c-Myc. Therefore, our results suggest that 15d-PGJ(2) can block ERalpha function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ERalpha DBD, resulting in fundamental inhibition of both hormone-dependent and hormone-independent ERalpha transcriptional activity.

A serious obstacle to successful treatment of estrogen receptor (ER)-positive human breast cancer is cell resistance to tamoxifen (TAM) therapy. Here we show that the electrophile disulfide benzamide (DIBA), an ER zinc finger inhibitor, blocks ligand-dependent and -independent cell growth of TAM-resistant breast cancer in vitro and in vivo. Such inhibition depends on targeting disruption of the ER DNA-binding domain and its communication with neighboring functional domains, facilitating ERalpha dissociation from its coactivator AIB1 and concomitant association with its corepressor NCoR bound to chromatin. DIBA does not affect phosphorylation of HER2, MAPK, AKT, and AIB1, suggesting that DIBA-modified ERalpha may induce a switch from agonistic to antagonistic effects of TAM on resistant breast cancer cells.

Multiple myeloma (MM) has prominent features of karyotypic instability at the earliest stage, leading to extreme genetic abnormalities as the disease progresses. These successive genetic alterations can be attributed, in part, to defects in DNA repair pathways. A possible mechanism of dysregulation of the DNA repair pathway is epigenetic gene silencing. Therefore, we sought to determine the methylation status of enzymes involved in the base excision repair pathway in multiple myeloma cell lines. Here, we report the aberrant DNA methylation of TDG, one of the enzymes involved in base excision repair of damaged DNA, in several multiple myeloma cell lines but not in normal human plasma cells. DNA hypermethylation of TDG in the MM cell lines leads to lower gene expression levels that results in less efficient DNA repair activity in response to hydrogen peroxideinduced DNA damage. Expression of exogenous TDG can functionally compensate for lower repair activities of damaged DNA in the KAS-6/1 myeloma cell line, which has extensive DNA hypermethylation of the TDG promoter. Hypermethylation of DNA damage repair genes in MM cell lines may provide an explanation for the frequent genomic instability, as well as point mutations, that are encountered in MM.

Autocrine pathways of proliferative and anti-apoptotic growth factors represent a serious impediment to the treatment of many types of tumors. In particular, interleukin-6 (IL-6), a pleiotropic cytokine known to play a critical role in the survival and growth of multiple myeloma cells, participates in an autocrine stimulation loop that serves to inhibit the induction of apoptosis during chemotherapy. Manganese superoxide dismutase (MnSOD) is an important antioxidant enzyme encoded by the SOD2 gene that attenuates oxidative free radicals in the mitochondria by catalyzing the formation of hydrogen peroxide from superoxide radicals. Transcription factor activity and binding is influenced by the oxidative state of cells, and dysregulation of MnSOD levels can result in abnormal patterns of gene expression. In the human multiple myeloma cell line IM-9, an autocrine IL-6 loop exists, which enables the cell to resist the effects of dexamethasone, a common treatment for multiple myeloma. Here, we show that SOD2 expression is epigenetically silenced in IM-9 cells, and replacement of MnSOD reduces cell proliferation and partially restores susceptibility to dexamethasone. The restoration of MnSOD also serves to decrease the expression levels of IL-6 by reducing the ability of activator protein-1, an important mediator of IL-6 expression in multiple myeloma cells, to bind to its enhancer site. These results show the importance of free radical-mediated dysregulation of autocrine growth factor loops in tumor cells and their effect on cell growth and response to chemotherapy.

A strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. Interleukin 6 (IL-6) is an inflammatory cytokine known to play a role in the growth and survival of many types of tumors, yet the mechanisms employed by this pleomorphic cytokine to accomplish this feat are still poorly understood. Another important factor in tumor development seems to be the hypermethylation of CpG islands located within the promoter regions of tumor suppressor genes. This common epigenetic alteration enables tumor cells to reduce or inactivate the expression of important tumor suppressor and cell cycle regulatory genes. Here we show that in the IL-6-responsive human multiple myeloma cell line KAS 6/1, the promoter region of p53 is epigenetically modified by methyltransferases, resulting in decreased levels of expression. Furthermore, cells treated with IL-6 exhibit an increase in the expression of the DNA maintenance methylation enzyme, DNMT-1. The DNA methyltransferase inhibitor zebularine reverses the methylation of the p53 promoter, allowing the resumption of its expression. However, when zebularine is withdrawn from the cells, the reestablishment of the original CpG island methylation within the p53 promoter does not occur in the absence of IL-6, and cells which do not receive IL-6 eventually die, as p53 expression continues unchecked by remethylation. Interestingly, this loss of viability seems to involve not the withdrawal of cytokine, but the inability of the cell to resilence the promoter. Consistent with this model, when cells that express IL-6 in an autocrine fashion are subjected to identical treatment, p53 expression is reduced shortly after withdrawal of zebularine. Therefore, it seems IL-6 is capable of maintaining promoter methylation thus representing one of the possible mechanisms used by inflammatory mediators in the growth and survival of tumors.

During tumorigenesis, selective proliferative advantage in certain cell subsets is associated with accumulation of multiple genetic alterations. For instance, multiple myeloma is characterized by frequent karyotypic instability at the earliest stage, progressing to extreme genetic abnormalities as the disease progresses. These successive genetic alterations can be attributed, in part, to defects in DNA repair pathways, perhaps based on epigenetic gene silencing of proteins involved in DNA damage repair. Here we report epigenetic hypermethylation of the hHR23B gene, a key component of the nucleotide excision repair in response to DNA damage, in interleukin-6 (IL-6)-responsive myeloma KAS-6/1 cells. This hypermethylation was significantly abated by Zebularine, a potent demethylating agent, with a consequent increase in the hHR23B mRNA level. Subsequent removal of this drug and supplementation with IL-6 in the culture medium re-established DNA hypermethylation of the hHR23B gene and silencing of mRNA expression levels. The inclination of DNA to be remethylated, at least within the hHR23B gene promoter region, reflects an epigenetic driving force by the cytogenetic/tumorigenic status of KAS-6/1 myeloma. The IL-6 response of KAS-6/1 myeloma also raises a question of whether the proneoplastic growth factor, such as IL-6, supports the epigenetic silencing of important DNA repair genes via promoter hypermethylation during the development of multiple myeloma.

Cytosine methylation, an epigenetic form of regulating gene transcription, has gained importance upon the discovery that genes involved in the carcinogenic process may be regulated by this mechanism and, moreover, that certain cancers respond to treatment with demethylation-promoting drugs. Typically, the use of DNA methyltransferase inhibitor drugs results in the up-regulation of important tumor suppressor genes, previously down-regulated by the existence of abnormal cytosine methylation within their promoters. Here, we show microarray and RT-PCR results indicating that many genes are down-regulated upon treatment of KAS 6/1 multiple myeloma cells with Zebularine, a demethylating agent. Our findings suggest that, in addition to the typical methylation inhibitor-induced up-regulation of genes, removal of methylation in some genes may have a profound down-regulating effect upon their expression. The analysis of gene function showed that, of the down-regulated genes, 38 are associated with cell proliferation and/or cancer. Our analysis of the promoters of the subset of selected genes containing CpG islands showed that the distribution of cis elements differs between genes up- and down-regulated by methylation. Finally, we propose a model which shows how genes containing methylation sites within their basic promoters and/or enhancer sequences are susceptible to down-regulation, whereas genes methylated within silencer regions are up-regulated, thus providing a model as to how DNA methylation could induce such opposing effects on transcription., (Copyright© 2004 International Institute of Anticaner Research (Dr. John G. Delinassios), All rights reserved.)

The epigenetic control of gene transcription in cancer has been the theme of many recent studies and therapeutic approaches. Carcinogenesis is frequently associated with hypermethylation and consequent down-regulation of genes that prevent cancer, e.g., those that control cell proliferation and apoptosis. We used the demethylating drug zebularine to induce changes in DNA methylation, then examined patterns of gene expression using cDNA array analysis and Restriction Landmark Genomic Scanning followed by RNase protection assay and reverse transcription-PCR to confirm the results. Microarray studies revealed that many genes were epigenetically regulated by methylation. We concluded that methylation decreased the expression of, or silenced, several genes, contributing to the growth and survival of multiple myeloma cells. For example, a number of genes (BAD, BAK, BIK, and BAX) involved in apoptosis were found to be suppressed by methylation. Sequenced methylation-regulated DNA fragments identified by Restriction Landmark Genomic Scanning were found to contain CpG islands, and some corresponded to promoters of genes that were regulated by methylation. We also observed that after the removal of the demethylating drug, the addition of interleukin 6 restored CpG methylation and re-established previously silenced gene patterns, thus implicating a novel role of interleukin 6 in processes regulating epigenetic gene repression and carcinogenesis.

Multiple myeloma (MM) remains largely incurable despite conventional and high-dose therapies. Therefore, novel biologically based treatment approaches are urgently required. Here we demonstrate that expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in MM cells and its agonists 15-d-PGJ2 and troglitazone completely abolished IL-6-inducible MM cell proliferation and induced apoptosis through affecting expression of multiple cell cycle or apoptosis genes, whereas PPARgamma antagonist GW9662 and PPARalpha agonist WY14643 did not display this inhibitory effect. These PPARgamma agonists significantly inhibited DNA binding and transactivation of STAT3 bound to the promoter of target genes in chromatin, but did not affect the expression of IL-6 receptor and phosphorylation of JAK/STAT3, MAPK, and PI3K/Akt. Interestingly, although inactivation of STAT3 by PPARgamma agonists is in a PPARgamma-dependent manner, the molecular mechanism by which two structurally distinct PPARgamma agonists suppress IL-6-activated STAT3 shows the divergent interactions between PPARgamma and STAT3 including direct or SMRT-mediated association.

Current antiestrogen therapy for breast cancer is limited by the mixed estrogenic and antiestrogenic activity of selective estrogen receptor modulators. Here we show that the function of zinc fingers in the estrogen receptor DNA-binding domain (DBD) is susceptible to chemical inhibition by electrophilic disulfide benzamide and benzisothiazolone derivatives, which selectively block binding of the estrogen receptor to its responsive element and subsequent transcription. These compounds also significantly inhibit estrogen-stimulated cell proliferation, markedly reduce tumor mass in nude mice bearing human MCF-7 breast cancer xenografts, and interfere with cell-cycle and apoptosis regulatory gene expression. Functional assays and computational analysis support a molecular mechanism whereby electrophilic agents preferentially disrupt the vulnerable C-terminal zinc finger, thus suppressing estrogen receptor-mediated breast carcinoma progression. Our results provide the proof of principle for a new strategy to inhibit breast cancer at the level of DNA binding, rather than the classical antagonism of estrogen binding.

Breast cancer, the most common malignancy in women, has been demonstrated to be associated with the steroid hormone estrogen and its receptor (ER), a ligand-activated transcription factor. Therefore, we developed a phosphorothiolate cis-element decoy against the estrogen response element (ERE decoy) to target disruption of ER DNA binding and transcriptional activity. Here, we showed that the ERE decoy potently ablated the 17beta-estrogen-inducible cell proliferation and induced apoptosis of human breast carcinoma cells by functionally affecting expression of c-fos gene and AP-1 luciferase gene reporter activity. Specificity of the decoy was demonstrated by its ability to directly block ER binding to a cis-element probe and transactivation. Moreover, the decoy failed to inhibit ER-mediated mitogen-activated protein kinase signaling pathways and cell growth of ER-negative breast cancer cells. Taken together, these data suggest that estrogen-mediated cell growth of breast cancer cells can be preferentially restricted via targeted disruption of ER at the level of DNA binding by a novel and specific decoy strategy applied to steroid nuclear receptors.

RANTES (regulated on activation normal T cell expressed and secreted), a ligand for the CC chemokine receptor 5, potently inhibits HIV-1 replication in vitro. We tested the influence of four RANTES single nucleotide polymorphism (SNP) variants and their haplotypes on HIV-1 infection and AIDS progression in five AIDS cohorts. Three SNPs in the RANTES gene region on chromosome 17 (403A in the promoter, In1.1C in the first intron, and 3'222C in the 3' untranslated region) are associated with increased frequency of HIV-1 infection. The common In1.1C SNP allele is nested within an intronic regulatory sequence element that exhibits differential allele binding to nuclear proteins and a down-regulation of gene transcription. The In1.1C allele or haplotypes that include In1.1C display a strong dominant association with rapid progression to AIDS among HIV-1-infected individuals in African-American, European-American, and combined cohorts. The principal RANTES SNP genetic influence on AIDS progression derives from the down-regulating RANTES In1.1C allele, although linkage disequilibrium with adjoining RANTES SNPs including a weaker up-regulating RANTES promoter allele (-28G), can modify the observed epidemiological patterns. The In1.1C-bearing genotypes account for 37% of the attributable risk for rapid progression among African Americans and may also be an important influence on AIDS progression in Africa. The diminished transcription of RANTES afforded by the In1.1C regulatory allele is consistent with increased HIV-1 spread in vivo, leading to accelerated progression to AIDS.

STAT3, a member of signal transducers and activators of transcription (STATs) originally discovered as mediators in cytokine signaling pathways, plays an active role in oncogenesis. However, the function of STAT3 in signaling multistage carcinogenesis, especially in transformation of tumor-promotion sensitive epithelial cells has not been elucidated. The present study demonstrates that STAT3 is activated in interleukin-6 induced transformation in mouse skin epithelial cells. DNA binding and transcriptional activities of STAT3 were significantly increased by interleukin-6. This induced anchorage-independent transformation in tumor-promotion sensitive JB6 mouse skin P+ cells but not in the resistant variant P- cells. Two forms of dominant negative STAT3 (mutant of transcriptional domain, mF, or DNA-binding domain, mD) were stably transfected into P+ cells. Activation of STAT3 was abolished and importantly, interleukin-6 induced anchorage-independent growth was absent in both mutant STAT3 transfectants. To determine the genes targeted by STAT3, three matrix metalloproteinase proteins linked with carcinogenesis of epithelial cells were analysed. Both basal and interleukin-6 induced expression of collagenase I and stromelysin I, but not gelatinase A, were inhibited in the mutant STAT3 transfectants. Furthermore, transfection of a wild type STAT3 restored STAT3 transactivation and response to interleukin-6 induced transformation in mutant STAT3 transfectants, which up-regulated collagenase I and stromelysin I as well. Together, these results provide the first evidence that STAT3 activation is required in the progression of multistage carcinogenesis of mouse skin epithelial cells, and matrix metalloproteinases are actively involved in STAT3-mediated cell transformation.

The respective development of either T helper type 1 (Th1) or Th2 cells is believed to be mediated by the effects of cytokines acting directly on Th precursors (Thp). We have generated evidence for an indirect monocyte-dependent immunoregulatory pathway. Recently, interleukin (IL) 4 has been shown to produce "new" potential peroxisome proliferator-activated receptor gamma (PPARgamma) ligands by inducing macrophage 12/15-lipoxygenase (12/15-LO). We have shown previously that the activated PPARgamma is a profound inhibitor of IL-2 transcription in human T lymphocytes. It is hypothetically possible that IL-4 might indirectly affect IL-2 production by Thp cells via macrophage-derived PPARgamma ligands. Using human monocytes and T lymphocytes from same donors, we have found that monocyte 12/15-LO products mediate the indirect inhibitory effect of IL-4 on anti-CD3- or phytohemagglutinin/phorbol 12-myristate 13-acetate-stimulated IL-2 production by T lymphocytes. We further analyzed which major 12/15-LO metabolites contributed to the above inhibition. 13-Hydroxyoctadecadienoic acid (13-HODE), a 12/15-LO product, markedly blocked IL-2 production by human blood T lymphocytes, but not Jurkat T cells. Moreover, the IL-4-conditioned macrophage medium contained a sufficient amount of 13-HODE and anti-13-HODE antibody indeed neutralized the inhibitory effects of the IL-4-conditional medium on T-cell IL-2 production. Using human T lymphocytes and the PPARgamma-transfected Jurkat T cells, we demonstrated the specific inhibition by 13-HODE of the transcription factors NFAT (nuclear factor of activated T cells) and nuclear factor kappaB, the IL-2 promoter reporter, and IL-2 production. However, 15-hydroxytetraenoic acid had little inhibitory effect. The potency of such inhibitory effects correlates well with the capability of the above metabolic lipids to activate PPARgamma. These data provide a mechanism whereby IL-4 may indirectly affect Thp function via PPARgamma activated by macrophage products of the 12/15-LO pathway.

Methylation of mammalian DNA by the DNA methyltransferase enzyme (dnmt-1) at CpG dinucleotide sequences has been recognized as an important epigenetic control mechanism in regulating the expression of cellular genes (Yen, R. W., Vertino, P. M., Nelkin, B. D., Yu, J. J., el-Deiry, W., Cumaraswamy, A., Lennon, G. G., Trask, B. J., Celano, P., and Baylin, S. B. (1992) Nucleic Acids Res. 20, 2287-2291; Ramchandani, S., Bigey, P., and Szyf, M. (1998) Biol. Chem. 379, 535-5401). Here we show that interleukin (IL)-6 regulates the methyltransferase promoter and resulting enzyme activity, which requires transcriptional activation by the Fli-1 transcription factor (Spyropoulos, D. D., Pharr, P. N., Lavenburg, K. R., Jackers, P., Papas, T. S., Ogawa, M., and Watson, D. K. (1998) Mol. Cell. Biol. 15, 5643-5652). The data suggest that inflammatory cytokines such as IL-6 may exert many epigenetic changes in cells via the regulation of the methyltransferase gene. Furthermore, IL-6 regulation of transcription factors like Fli-1, which can help to direct cells along opposing differentiation pathways, may in fact be reflected in part by their ability to regulate the methylation of cellular genes.