Epigenetic Effects of Resveratrol on Oncogenic Signaling in Breast Cancer.

The crosstalk between oncogenic signaling pathways plays a crucial role in driving cancer development. We previously demonstrated that dietary polyphenols, specifically resveratrol (RSV) and other stilbenoids, epigenetically target oncogenes for silencing via DNA hypermethylation in breast cancer. In the present study, we identify signal transduction regulators among RSV-hypermethylated targets and investigate the functional role of RSV-mediated DNA hypermethylation in the regulation of Hedgehog and Wnt signaling. Non-invasive ER-positive MCF-7 and highly invasive triple-negative MCF10CA1a human breast cancer cell lines were used as experimental models. Upon 9-day exposure to 15 µM RSV, pyrosequencing and qRT-PCR were performed to assess DNA methylation and expression of GLI2 and WNT4, which are upstream regulators of the Hedgehog and Wnt pathways, respectively. Our results showed that RSV led to a DNA methylation increase within GLI2 and WNT4 enhancers, which was accompanied by decreases in gene expression. Consistently, we observed the downregulation of genes downstream of the Hedgehog and Wnt signaling, including common targets shared by both pathways, CCND1 and CYR61. Further analysis using chromatin immunoprecipitation identified increased H3K27 trimethylation and decreased H3K9 and H3K27 acetylation, along with abolishing OCT1 transcription factor binding. Those changes indicate a transcriptionally silent chromatin state at GLI2 and WNT4 enhancers. The inhibition of the Wnt signal transduction was confirmed using a phospho-antibody array that demonstrated suppression of positive and stimulation of negative Wnt regulators. In conclusion, our results provide scientific evidence for dietary polyphenols as epigenetics-modulating agents that act to re-methylate and silence oncogenes, reducing the oncogenic signal transduction. Targeting such an action could be an effective strategy in breast cancer prevention and/or adjuvant therapy.

Epigenetic aberrations of gene expression in a rat model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is mostly triggered by environmental and life-style factors and may involve epigenetic aberrations. However, a comprehensive documentation of the link between the dysregulated epigenome, transcriptome, and liver carcinogenesis is lacking. In the present study, Fischer-344 rats were fed a choline-deficient (CDAA, cancer group) or choline-sufficient (CSAA, healthy group) L-amino acid-defined diet. At the end of 52 weeks, transcriptomic alterations in livers of rats with HCC tumours and healthy livers were investigated by RNA sequencing. DNA methylation and gene expression were assessed by pyrosequencing and quantitative reverse-transcription PCR (qRT-PCR), respectively. We discovered 1,848 genes that were significantly differentially expressed in livers of rats with HCC tumours (CDAA) as compared with healthy livers (CSAA). Upregulated genes in the CDAA group were associated with cancer-related functions, whereas macronutrient metabolic processes were enriched by downregulated genes. Changes of highest magnitude were detected in numerous upregulated genes that govern key oncogenic signalling pathways, including Notch, Wnt, Hedgehog, and extracellular matrix degradation. We further detected perturbations in DNA methylating and demethylating enzymes, which was reflected in decreased global DNA methylation and increased global DNA hydroxymethylation. Four selected upregulated candidates, Mmp12, Jag1, Wnt4, and Smo, demonstrated promoter hypomethylation with the most profound decrease in Mmp12. MMP12 was also strongly overexpressed and hypomethylated in human HCC HepG2 cells as compared with primary hepatocytes, which coincided with binding of Ten-eleven translocation 1 (TET1). Our findings provide comprehensive evidence for gene expression changes and dysregulated epigenome in HCC pathogenesis, potentially revealing novel targets for HCC prevention/treatment.

Pterostilbene Changes Epigenetic Marks at Enhancer Regions of Oncogenes in Breast Cancer Cells.

Epigenetic aberrations are linked to sporadic breast cancer. Interestingly, certain dietary polyphenols with anti-cancer effects, such as pterostilbene (PTS), have been shown to regulate gene expression by altering epigenetic patterns. Our group has proposed the involvement of DNA methylation and DNA methyltransferase 3B (DNMT3B) as vital players in PTS-mediated suppression of candidate oncogenes and suggested a role of enhancers as target regions. In the present study, we assess a genome-wide impact of PTS on epigenetic marks at enhancers in highly invasive MCF10CA1a breast cancer cells. Following chromatin immunoprecipitation (ChIP)-sequencing in MCF10CA1a cells treated with 7 µM PTS for 9 days, we discovered that PTS leads to increased binding of DNMT3B at enhancers of 77 genes, and 17 of those genes display an overlapping decrease in the occupancy of trimethylation at lysine 36 of histone 3 (H3K36me3), a mark of active enhancers. We selected two genes, PITPNC1 and LINC00910, and found that their enhancers are hypermethylated in response to PTS. These changes coincided with the downregulation of gene expression. Of importance, we showed that 6 out of 17 target enhancers, including PITPNC1 and LINC00910, are bound by an oncogenic transcription factor OCT1 in MCF10CA1a cells. Indeed, the six enhancers corresponded to genes with established or putative cancer-driving functions. PTS led to a decrease in OCT1 binding at those enhancers, and OCT1 depletion resulted in PITPNC1 and LINC00910 downregulation, further demonstrating a role for OCT1 in transcriptional regulation. Our findings provide novel evidence for the epigenetic regulation of enhancer regions by dietary polyphenols in breast cancer cells.

Pterostilbene leads to DNMT3B-mediated DNA methylation and silencing of OCT1-targeted oncogenes in breast cancer cells.

Transcription factor (TF)-mediated regulation of genes is often disrupted during carcinogenesis. The DNA methylation state of TF-binding sites may dictate transcriptional activity of corresponding genes. Stilbenoid polyphenols, such as pterostilbene (PTS), have been shown to exert anticancer action by remodeling DNA methylation and gene expression. However, the mechanisms behind these effects still remain unclear. Here, the dynamics between oncogenic TF OCT1 binding and de novo DNA methyltransferase DNMT3B binding in PTS-treated MCF10CA1a invasive breast cancer cells has been explored. Using chromatin immunoprecipitation (ChIP) followed by next generation sequencing, we determined 47 gene regulatory regions with decreased OCT1 binding and enriched DNMT3B binding in response to PTS. Most of those genes were found to have oncogenic functions. We selected three candidates, PRKCA, TNNT2, and DANT2, for further mechanistic investigation taking into account PRKCA functional and regulatory connection with numerous cancer-driving processes and pathways, and some of the highest increase in DNMT3B occupancy within TNNT2 and DANT2 enhancers. PTS led to DNMT3B recruitment within PRKCA, TNNT2, and DANT2 at loci that also displayed reduced OCT1 binding. Substantial decrease in OCT1 with increased DNMT3B binding was accompanied by PRKCA promoter and TNNT2 and DANT2 enhancer hypermethylation, and gene silencing. Interestingly, DNA hypermethylation of the genes was not detected in response to PTS in DNMT3B-CRISPR knockout MCF10CA1a breast cancer cells. It indicates DNMT3B-dependent methylation of PRKCA, TNNT2, and DANT2 upon PTS. Our findings provide a better understanding of mechanistic players and their gene targets that possibly contribute to the anticancer action of stilbenoid polyphenols.

Curcumin from Turmeric Rhizome: A Potential Modulator of DNA Methylation Machinery in Breast Cancer Inhibition.

One of the most systematically studied bioactive nutraceuticals for its benefits in the management of various diseases is the turmeric-derived compounds: curcumin. Turmeric obtained from the rhizome of a perennial herb Curcuma longa L. is a condiment commonly used in our diet. Curcumin is well known for its potential role in inhibiting cancer by targeting epigenetic machinery, with DNA methylation at the forefront. The dynamic DNA methylation processes serve as an adaptive mechanism to a wide variety of environmental factors, including diet. Every healthy tissue has a precise DNA methylation pattern that changes during cancer development, forming a cancer-specific design. Hypermethylation of tumor suppressor genes, global DNA demethylation, and promoter hypomethylation of oncogenes and prometastatic genes are hallmarks of nearly all types of cancer, including breast cancer. Curcumin has been shown to modulate epigenetic events that are dysregulated in cancer cells and possess the potential to prevent cancer or enhance the effects of conventional anti-cancer therapy. Although mechanisms underlying curcumin-mediated changes in the epigenome remain to be fully elucidated, the mode of action targeting both hypermethylated and hypomethylated genes in cancer is promising for cancer chemoprevention. This review provides a comprehensive discussion of potential epigenetic mechanisms of curcumin in reversing altered patterns of DNA methylation in breast cancer that is the most commonly diagnosed cancer and the leading cause of cancer death among females worldwide. Insight into the other bioactive components of turmeric rhizome as potential epigenetic modifiers has been indicated as well.

The effects of clofarabine in ALL inhibition through DNA methylation regulation.

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF), a second-generation 2'-deoxyadenosine analog, possesses manifold anti-cancer activities. Our previous reports and some of others demonstrate the potential capacity of ClF to regulate the epigenetic machinery. The study presented here is the first to investigate the influence of ClF on modulators of the DNA methylation machinery, including DNMT1 and CDKN1A, in acute lymphoblastic leukemia (ALL) cells. ClF effects on promoter methylation and transcriptional activity of hypermethylated and silenced tumor suppressor genes (TSGs), including APC, CDKN2A, PTEN, and RARB, have been tested as well. Methylation level of the proximal promoter region of APC, CDKN2A, PTEN, and RARB, as well as expression of those TSGs, DNMT1 and CDKN1A, were estimated by using a methylation-sensitive restriction analysis and qPCR, respectively. The Nalm-6 cell line was used as an experimental in vitro model of ALL cells. We observed ClF-mediated inhibition of cellular viability and apoptosis induction of Nalm-6 cells with an increased percentage of cells positive for active Caspase-3. Interestingly, exposure of Nalm-6 cells to CIF at 20 nM concentration for three days has led to a significant DNMT1 downregulation, accompanied by robust CDKN1A upregulation. ClF caused hypomethylation of APC, CDKN2A, and PTEN, with a concomitant increase in their transcript levels. Taken together, our results demonstrate the ability of ClF to reactivate DNA methylation-silenced TSGs in ALL cells. This may implicate translational significance of our findings and support ClF application as a new epigenetic modulator in the anti-leukemia therapy.

Dietary antioxidants remodel DNA methylation patterns in chronic disease.

Chronic diseases account for over 60% of all deaths worldwide according to the World Health Organization reports. Majority of cases are triggered by environmental exposures that lead to aberrant changes in the epigenome, specifically, the DNA methylation patterns. These changes result in altered expression of gene networks and activity of signalling pathways. Dietary antioxidants, including catechins, flavonoids, anthocyanins, stilbenes and carotenoids, demonstrate benefits in the prevention and/or support of therapy in chronic diseases. This review provides a comprehensive discussion of potential epigenetic mechanisms of antioxidant compounds in reversing altered patterns of DNA methylation in chronic disease. Antioxidants remodel the DNA methylation patterns through multiple mechanisms, including regulation of epigenetic enzymes and chromatin remodelling complexes. These effects can further contribute to antioxidant properties of the compounds. On the other hand, decrease in oxidative stress itself can impact DNA methylation delivering additional link between antioxidant mechanisms and epigenetic effects of the compounds. LINKED ARTICLES: This article is part of a themed section on The Pharmacology of Nutraceuticals. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.6/issuetoc.

Clofarabine­phytochemical combination exposures in CML cells inhibit DNA methylation machinery, upregulate tumor suppressor genes and promote caspase­dependent apoptosis.

Clofarabine (2­chloro­2'­fluoro­2'­deoxyarabinosyladenine, CIF), a second­generation 2'­deoxyadenosine analog, possesses a variety of anti­cancer activities, including the capacity to modulate DNA methylation marks. Bioactive nutrients, including resveratrol (RSV) and all­trans retinoic acid (ATRA) have been indicated to regulate epigenetic machinery in malignant cells. The purpose of the current study was to evaluate whether the tested phytochemicals, RSV or ATRA, can improve the therapeutic epigenetic effects of CIF in chronic myeloid leukemia (CML) cells. The present study investigates, to the best of our knowledge, for the first time, the influence of CIF in combination with RSV or ATRA on the expression of relevant modifiers of DNA methylation machinery, including DNA Methyltransferase 1 (DNMT1) and Cyclin dependent kinase inhibitor 1A (CDKN1A) in CML cells. Subsequently, the combinatorial effects on promoter methylation and transcript levels of methylation­silenced tumor suppressor genes (TSGs), including phosphatase and tensin homologue (PTEN) and retinoic acid receptor beta (RARB), were estimated using MSRA and qPCR, respectively. The tested TSGs were chosen according to bioinformatical analysis of publicly available clinical data of human DNA methylation and gene expression arrays in leukemia patients. The K562 cell line was used as an experimental CML in vitro model. Following a period of 72 h exposure of K562 cells, the tested combinations led to significant cell growth inhibition and induction of caspase­3­dependent apoptosis. These observations were accompanied by DNMT1 downregulation and CDKN1A upregulation, with a concomitant enhanced decrease in DNMT1 protein level, especially after ATRA treatment with CIF. Concurrent methylation­mediated RARB and PTEN reactivation was detected. The results of the current study demonstrated that CIF that was used in combination with the tested phytochemicals, RSV or ATRA, exhibited a greater ability to remodel DNA methylation marks and promote cell death in CML cells. These results may support the application of CIF combinations with natural bioactive agents in anti­leukemic epigenetic therapy.

Stilbenoid-Mediated Epigenetic Activation of Semaphorin 3A in Breast Cancer Cells Involves Changes in Dynamic Interactions of DNA with DNMT3A and NF1C Transcription Factor.

SCOPE: Loci-specific increase in DNA methylation occurs in cancer and may underlie gene silencing. It is investigated whether dietary stilbenoids, resveratrol, and pterostilbene exert time-dependent effects on DNA methylation patterns and specifically methylation-silenced tumor suppressor genes in breast cancer cells. METHODS AND RESULTS: Following genome-wide DNA methylation analysis with Illumina-450K, changes characteristic of early and late response to stilbenoids are identified. Interestingly, often the same genes but at different CpG loci, the same gene families, or the same functional gene categories are affected. CpG loci that lose methylation in exposed cells correspond to genes functionally associated with cancer suppression. There is a group of genes, including SEMA3A, at which the magnitude of hypomethylation in response to stilbenoids rises with increasing invasive potential of cancer cells. Decreased DNA methylation at SEMA3A promoter and concomitant gene upregulation coincide with increased occupancy of active histone marks. Open chromatin upon exposure to stilbenoids may be linked to decreased DNMT3A binding followed by increased NF1C transcription factor occupancy. Sequestration of DNMT3A is possibly a result of stilbenoid-mediated increase in SALL3 expression, which was previously shown to bind and inhibit DNMT3A activity. CONCLUSIONS: The findings define mechanistic players in stilbenoid-mediated epigenetic reactivation of genes suppressing cancer.

Novel Clofarabine-Based Combinations with Polyphenols Epigenetically Reactivate Retinoic Acid Receptor Beta, Inhibit Cell Growth, and Induce Apoptosis of Breast Cancer Cells.

An epigenetic component, especially aberrant DNA methylation pattern, has been shown to be frequently involved in sporadic breast cancer development. A growing body of literature demonstrates that combination of agents, i.e. nucleoside analogues with dietary phytochemicals, may provide enhanced therapeutic effects in epigenetic reprogramming of cancer cells. Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF), a second-generation 2'-deoxyadenosine analogue, has numerous anti-cancer effects, including potential capacity to regulate epigenetic processes. Our present study is the first to investigate the combinatorial effects of ClF (used at IC50 concentration) with epigallocatechin-3-gallate (EGCG, tea catechin) or genistein (soy phytoestrogen), at physiological concentrations, on breast cancer cell growth, apoptosis, and epigenetic regulation of retinoic acid receptor beta (RARB) transcriptional activity. In MCF7 and MDA-MB-231 cells, RARB promoter methylation and expression of RARB, modifiers of DNA methylation reaction (DNMT1, CDKN1A, TP53), and potential regulator of RARB transcription, PTEN, were estimated using methylation-sensitive restriction analysis (MSRA) and quantitative real-time polymerase chain reaction (qPCR), respectively. The combinatorial exposures synergistically or additively inhibited the growth and induced apoptosis of breast cancer cells, followed by RARB hypomethylation with concomitant multiple increase in RARB, PTEN, and CDKN1A transcript levels. Taken together, our results demonstrate the ability of ClF-based combinations with polyphenols to promote cancer cell death and reactivate DNA methylation-silenced tumor suppressor genes in breast cancer cells with different invasive potential.

Loci-specific differences in blood DNA methylation in HBV-negative populations at risk for hepatocellular carcinoma development.

Late onset of clinical symptoms in hepatocellular carcinoma (HCC) results in late diagnosis and poor disease outcome. Approximately 85% of individuals with HCC have underlying liver cirrhosis. However, not all cirrhotic patients develop cancer. Reliable tools that would distinguish cirrhotic patients who will develop cancer from those who will not are urgently needed. We used the Illumina HumanMethylation450 BeadChip microarray to test whether white blood cell DNA, an easily accessible source of DNA, exhibits site-specific changes in DNA methylation in blood of diagnosed HCC patients (post-diagnostic, 24 cases, 24 controls) and in prospectively collected blood specimens of HCC patients who were cancer-free at blood collection (pre-diagnostic, 21 cases, 21 controls). Out of 22 differentially methylated loci selected for validation by pyrosequencing, 19 loci with neighbouring CpG sites (probes) were confirmed in the pre-diagnostic study group and subjected to verification in a prospective cirrhotic cohort (13 cases, 23 controls). We established for the first time 9 probes that could distinguish HBV-negative cirrhotic patients who subsequently developed HCC from those who stayed cancer-free. These probes were identified within regulatory regions of BARD1, MAGEB3, BRUNOL5, FXYD6, TET1, TSPAN5, DPPA5, KIAA1210, and LSP1. Methylation levels within DPPA5, KIAA1210, and LSP1 were higher in prospective samples from HCC cases vs. cirrhotic controls. The remaining probes were hypomethylated in cases compared with controls. Using blood as a minimally invasive material and pyrosequencing as a straightforward quantitative method, the established probes have potential to be developed into a routine clinical test after validation in larger cohorts.

Subtle Alterations in DNA Methylation Patterns in Normal Cells in Response to Dietary Stilbenoids.

SCOPE: Searching for correlations between dietary polyphenols and risk of chronic diseases has been a challenge due to the lack of quantitative evaluation methods of long-term exposure. We previously observed substantial DNA methylation changes in human cancer cells upon treatment with polyphenols of the stilbenoid class. When induced in normal cells, such molecular changes may persist and reflect chronic exposure. METHODS AND RESULTS: Illumina 450K microarray is used to delineate a genome wide DNA methylation landscape in MCF10A human immortalized mammary epithelial cells exposed to resveratrol (RSV) at noncytotoxic 15 µM dose for 9 days. Subtle alterations are observed suggesting remodeling of DNA methylation patterns rather than switch on/off changes. Using pyrosequencing, DNA methylation is quantitatively measured at eight CpG sites located within KCNJ4, RNF169, BCHE, DAOA, HOXA9, RUNX3, KRTAP2-1, and TAGAP, upon exposure to RSV or pterostilbene and shows similar differences induced by both stilbenoids. Two of the probes, Runx3 and Kcnj4, are successfully verified in whole blood DNA from healthy rats on diets supplemented with stilbenoids. CONCLUSIONS: The study provides strong support for testing the utility of polyphenol-mediated changes in DNA methylation as quantitative measures of long-term dietary exposures in nutritional epidemiology and clinical trials.

Inhibition of breast cancer cell growth by the combination of clofarabine and sulforaphane involves epigenetically mediated CDKN2A upregulation.

Many antineoplastic nucleoside analogue-based combinatorial strategies focused on remodelling aberrant DNA methylation patterns have been developed. The number of studies demonstrate high efficacy of bioactive phytochemicals in support of conventional chemotherapy. Our recent discoveries of the epigenetic effects of clofarabine (2'-deoxyadenosine analogue, antileukaemic drug) and clofarabine-based combinations with dietary bioactive compounds in breast cancer cells led us to look for more DNA methylation targets of these cancer-preventive agents. In the present study, using methylation-sensitive restriction analysis (MSRA) and qPCR, we showed that clofarabine in combination with sulforaphane, a phytochemical from cruciferous vegetables, significantly reactivates DNA methylation-silenced CDKN2A tumour suppressor and inhibits cancer cell growth at a non-invasive breast cancer stage.

CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter.

DNA hypermethylation at the promoter of tumour-suppressor genes is tightly correlated with their transcriptional repression and recognized as the hallmark of majority of cancers. Epigenetic silencing of tumour suppressor genes impairs their cellular functions and activates a cascade of events driving cell transformation and cancer progression. Here, we examine site-specific and spatiotemporal alteration in DNA methylation at a target region in BRCA1 gene promoter, a model tumour suppressor gene. We have developed a programmable CRISPR-Cas9 based demethylase tool containing the deactivated Cas9 (dCas9) fused to the catalytic domain (CD) of Ten-Eleven Translocation (TET) dioxygenase1 (TET1CD). The fusion protein selectively demethylates targeted regions within BRCA1 promoter as directed by the designed single-guide RNAs (sgRNA), leading to the transcriptional up-regulation of the gene. We also noticed the increment in 5-hydroxymethylation content (5-hmC) at the target DNA site undergoing the most profound demethylation. It confirms the catalytic activity of TET1 in TET1-dCas9 fusion proteins-mediated demethylation at these target sequences. The modular design of the fusion constructs presented here allows for the selective substitution of other chromatin or DNA modifying enzymes and for loci-specific targeting to uncover epigenetic regulatory pathways at gene promoters and other selected genomic regions.

Stilbenoids remodel the DNA methylation patterns in breast cancer cells and inhibit oncogenic NOTCH signaling through epigenetic regulation of MAML2 transcriptional activity.

DNA hypomethylation was previously implicated in cancer progression and metastasis. The purpose of this study was to examine whether stilbenoids, resveratrol and pterostilbene thought to exert anticancer effects, target genes with oncogenic function for de novo methylation and silencing, leading to inactivation of related signaling pathways. Following Illumina 450K, genome-wide DNA methylation analysis reveals that stilbenoids alter DNA methylation patterns in breast cancer cells. On average, 75% of differentially methylated genes have increased methylation, and these genes are enriched for oncogenic functions, including NOTCH signaling pathway. MAML2, a coactivator of NOTCH targets, is methylated at the enhancer region and transcriptionally silenced in response to stilbenoids, possibly explaining the downregulation of NOTCH target genes. The increased DNA methylation at MAML2 enhancer coincides with increased occupancy of repressive histone marks and decrease in activating marks. This condensed chromatin structure is associated with binding of DNMT3B and decreased occupancy of OCT1 transcription factor at MAML2 enhancer, suggesting a role of DNMT3B in increasing methylation of MAML2 after stilbenoid treatment. Our results deliver a novel insight into epigenetic regulation of oncogenic signals in cancer and provide support for epigenetic-targeting strategies as an effective anticancer approach.

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine)--biochemical aspects of anticancer activity.

Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine) is a second generation analogue of 2'-deoxyadenosine connecting biochemical activities of its prototypes: cladribine (2-chloro-2'-deoxyadenosine) and fludarabine (2-fluoro-arabinosyladenine). This new anticancer drug is more effective (in low doses) and indicates higher oral bioavailability in comparison to its congeners. The studies indicated that the molecular mechanism of clofarabine cytotoxic action includes cell apoptosis, which results from inhibition (by the drug triphosphate nucleotides) of ribonucleotide reductase and DNA polymerases. The most recent research demonstrated also that action of the drug may cause up-expression of some genes on mRNA and protein levels. Clofarabine was synthesized in 1992 and in 2004 was approved for treatment of pediatric patients with refractory or relapsed acute lymphoblastic leukemia (ALL). Encouraging results of clinical trials with clofarabine in acute leukemias inclined to present background knowledge about multidirectional biomolecular mechanism of its cytotoxicity.