Global analysis of H3K27me3 as an epigenetic marker in prostate cancer progression.

BACKGROUND: H3K27me3 histone marks shape the inhibition of gene transcription. In prostate cancer, the deregulation of H3K27me3 marks might play a role in prostate tumor progression. METHODS: We investigated genome-wide H3K27me3 histone methylation profile using chromatin immunoprecipitation (ChIP) and 2X400K promoter microarrays to identify differentially-enriched regions in biopsy samples from prostate cancer patients. H3K27me3 marks were assessed in 34 prostate tumors: 11 with Gleason score > 7 (GS > 7), 10 with Gleason score ≤ 7 (GS ≤ 7), and 13 morphologically normal prostate samples. RESULTS: Here, H3K27me3 profiling identified an average of 386 enriched-genes on promoter regions in healthy control group versus 545 genes in GS ≤ 7 and 748 genes in GS > 7 group. We then ran a factorial discriminant analysis (FDA) and compared the enriched genes in prostate-tumor biopsies and normal biopsies using ANOVA to identify significantly differentially-enriched genes. The analysis identified ALG5, EXOSC8, CBX1, GRID2, GRIN3B, ING3, MYO1D, NPHP3-AS1, MSH6, FBXO11, SND1, SPATS2, TENM4 and TRA2A genes. These genes are possibly associated with prostate cancer. Notably, the H3K27me3 histone mark emerged as a novel regulatory mechanism in poor-prognosis prostate cancer. CONCLUSIONS: Our findings point to epigenetic mark H3K27me3 as an important event in prostate carcinogenesis and progression. The results reported here provide new molecular insights into the pathogenesis of prostate cancer.

The association between histone 3 lysine 27 trimethylation (H3K27me3) and prostate cancer: relationship with clinicopathological parameters.

BACKGROUND: It is well established that genetic and epigenetic alterations are common events in prostate cancer, which may lead to aberrant expression of critical genes. The importance of epigenetic mechanisms in prostate cancer carcinogenesis is increasingly evident. In this study, the focus will be on histone modifications and the primary objectives are to map H3K27me3 marks and quantify RAR beta 2, ER alpha, SRC3, RGMA, PGR, and EZH2 gene expressions in prostate cancer tissues compared to normal tissues. In addition, a data analysis was made in connection with the clinicopathological parameters. METHODS: 71 normal specimens and 66 cancer prostate tissues were randomly selected in order to assess the proportion of the repressive H3K27me3 mark and gene expression. H3K27me3 level was evaluated by ChIP-qPCR and mRNA expression using RT-qPCR between prostate cancer and normal tissues. Subsequently, western-blotting was performed for protein detection. The analysis of variance (ANOVA) was performed, and Tukey's test was used to correct for multiple comparisons (p-value threshold of 0.05). The principal component analysis (PCA) and discriminant factorial analysis (DFA) were used to explore the association between H3K27me3 level and clinicopathological parameters. RESULTS: The study demonstrated that H3K27me3 level was significantly enriched at the RAR beta 2, ER alpha, PGR, and RGMA promoter regions in prostate cancer tissues compared to normal tissues. After stratification by clinicopathological parameters, the H3K27me3 level was positively correlated with Gleason score, PSA levels and clinical stages for RAR beta 2, ER alpha, PGR, and RGMA. High H3K27me3 mark was significantly associated with decreased RAR beta 2, ER alpha, PGR and RGMA gene expressions in prostate cancer sample compared to the normal one. Moreover, the results showed that mRNA level of EZH2, AR and SRC3 are upregulated in prostate cancer compared to normal prostate tissues and this correlates positively with Gleason score, PSA levels and clinical stages. Obviously, these observations were confirmed by protein level using western-blot. CONCLUSIONS: This data clearly demonstrated that H3K27me3 level correlated with aggressive tumor features. Also this study revealed that reverse correlation of RAR beta 2, ER alpha, PGR, and RGMA expressions with EZH2, SRC3, and AR expressions in prostate cancer tissues suggests that these genes are the target of EZH2. Therefore, all therapeutic strategies leading to histone demethylation with epigenetic drugs such as histone methyltransferase inhibitor may be relevant treatments against prostate cancer.

SIRT1-dependent epigenetic regulation of H3 and H4 histone acetylation in human breast cancer.

Breast cancer is the most frequently diagnosed malignancy in women worldwide. It is well established that the complexity of carcinogenesis involves profound epigenetic deregulations that contribute to the tumorigenesis process. Deregulated H3 and H4 acetylated histone marks are amongst those alterations. Sirtuin-1 (SIRT1) is a class-III histone deacetylase deeply involved in apoptosis, genomic stability, gene expression regulation and breast tumorigenesis. However, the underlying molecular mechanism by which SIRT1 regulates H3 and H4 acetylated marks, and consequently cancer-related gene expression in breast cancer, remains uncharacterized. In this study, we elucidated SIRT1 epigenetic role and analyzed the link between the latter and histones H3 and H4 epigenetic marks in all 5 molecular subtypes of breast cancer. Using a cohort of 135 human breast tumors and their matched normal tissues, as well as 5 human-derived cell lines, we identified H3k4ac as a new prime target of SIRT1 in breast cancer. We also uncovered an inverse correlation between SIRT1 and the 3 epigenetic marks H3k4ac, H3k9ac and H4k16ac expression patterns. We showed that SIRT1 modulates the acetylation patterns of histones H3 and H4 in breast cancer. Moreover, SIRT1 regulates its H3 acetylated targets in a subtype-specific manner. Furthermore, SIRT1 siRNA-mediated knockdown increases histone acetylation levels at 6 breast cancer-related gene promoters: AR, BRCA1, ERS1, ERS2, EZH2 and EP300. In summary, this report characterizes for the first time the epigenetic behavior of SIRT1 in human breast carcinoma. These novel findings point to a potential use of SIRT1 as an epigenetic therapeutic target in breast cancer.

A new metabolic gene signature in prostate cancer regulated by JMJD3 and EZH2.

Histone methylation is essential for gene expression control. Trimethylated lysine 27 of histone 3 (H3K27me3) is controlled by the balance between the activities of JMJD3 demethylase and EZH2 methyltransferase. This epigenetic mark has been shown to be deregulated in prostate cancer, and evidence shows H3K27me3 enrichment on gene promoters in prostate cancer. To study the impact of this enrichment, a transcriptomic analysis with TaqMan Low Density Array (TLDA) of several genes was studied on prostate biopsies divided into three clinical grades: normal (n = 23) and two tumor groups that differed in their aggressiveness (Gleason score ≤ 7 (n = 20) and >7 (n = 19)). ANOVA demonstrated that expression of the gene set was upregulated in tumors and correlated with Gleason score, thus discriminating between the three clinical groups. Six genes involved in key cellular processes stood out: JMJD3, EZH2, MGMT, TRA2A, U2AF1 and RPS6KA2. Chromatin immunoprecipitation demonstrated collocation of EZH2 and JMJD3 on gene promoters that was dependent on disease stage. Gene set expression was also evaluated on prostate cancer cell lines (DU 145, PC-3 and LNCaP) treated with an inhibitor of JMJD3 (GSK-J4) or EZH2 (DZNeP) to study their involvement in gene regulation. Results showed a difference in GSK-J4 sensitivity under PTEN status of cell lines and an opposite gene expression profile according to androgen status of cells. In summary, our data describe the impacts of JMJD3 and EZH2 on a new gene signature involved in prostate cancer that may help identify diagnostic and therapeutic targets in prostate cancer.

TIP60: an actor in acetylation of H3K4 and tumor development in breast cancer.

AIM: The acetyltransferase TIP60 is reported to be downregulated in several cancers, in particular breast cancer, but the molecular mechanisms resulting from its alteration are still unclear. MATERIALS & METHODS: In breast tumors, H3K4ac enrichment and its link with TIP60 were evaluated by chromatin immunoprecipitation-qPCR and re-chromatin immunoprecipitation techniques. To assess the biological roles of TIP60 in breast cancer, two cell lines of breast cancer, MDA-MB-231 (ER-) and MCF-7 (ER+) were transfected with shRNA specifically targeting TIP60 and injected to athymic Balb-c mice. RESULTS: We identified a potential target of TIP60, H3K4. We show that an underexpression of TIP60 could contribute to a reduction of H3K4 acetylation in breast cancer. An increase in tumor development was noted in sh-TIP60 MDA-MB-231 xenografts and a slowdown of tumor growth in sh-TIP60 MCF-7 xenografts. CONCLUSION: This is evidence that the underexpression of TIP60 observed in breast cancer can promote the tumorigenesis of ER-negative tumors.

The Epigenetic Landscape of Promoter Genome-wide Analysis in Breast Cancer.

Breast cancer is a heterogeneous disease due to its clinico-pathological features and response to therapy. The classification of breast tumors based on their hormone receptor status and pathologic features. Post-translational histone modifications come into prominence for regulation of gene expression in cancer pathogenesis. Here, we analyzed dysregulation of H3K9ac and H3K27me3-enriched subtype-specific genes using ChIP-on-chip assay in breast cancer tumors and matched normal tissue samples. Breast cancer tumors were classified according to St Gallen Consensus 2013. Our results indicated that the promoter regions of genes modified by H3K9ac epi-mark are commonly associated with tumors with HER2-positive and TNBC subtype. H3K27me3-enriched genes were comprised of Luminal A and B1 subtypes. We constructed a network structure to elicit epigenetically regulated genes related with breast cancer progression. The central genes of the network (RUNX1, PAX3, GATA4 and DLX5) were subjected for epigenetically dysregulation in association with different breast cancer subtypes. Our study submits epigenetic mechanisms are crucial to elicit subtype-specific regulation in breast cancer and ChIP-on-chip assay provides a better understanding for breast tumorigenesis and new approaches for prevention and treatment.

Dual SIRT1 expression patterns strongly suggests its bivalent role in human breast cancer.

Breast cancer is the most common cancer in women, and the leading cause of cancer death in women worldwide. SIRT1 (silent mating type information regulation 2 homolog) 1 is a class-III histone deacetylase involved in apoptosis regulation, DNA repair and tumorigenesis. However, its role in breast carcinoma remains controversial, as both tumor-suppressive and tumor-promoting functions have been reported. Also, there are very few reports available where expression of SIRT1 is comprehensively analyzed in breast tumors classified by molecular subtype. Here, using a cohort of 50 human breast tumors and their matched normal tissues, we investigated SIRT1 expression levels in the 5 molecular subtypes of breast cancer according to the St Gallen classification (2013). Tumors and their corresponding normal tissue samples were collected from all patients, and SIRT1 mRNA and protein expression levels were then examined by real-time quantitative polymerase chain reaction and immunoblotting, respectively. After statistical analysis, the results showed a dual expression profile of SIRT1 in human breast carcinoma, with significant overexpression in luminal and HER2-enriched subtypes and significantly reduced expression in the triple-negative subtype. We also found an inverse correlation between SIRT1 expression and breast cancer aggressivity. These novel findings suggest that SIRT1 plays a dual role in breast tumors depending on its expression rate and the molecular subtype of the cancer. Our data also point to a potential role for SIRT1 as a prognostic biomarker in breast cancer.

High-throughput «Omics¼ technologies: New tools for the study of triple-negative breast cancer.

Triple negative breast cancer (TNBC) represents about 15% to 20% of all breast cancers and is typically associated with poorer outcome than other breast cancer subtypes. The heterogeneity of this breast cancer subtype and present lack of clinically established targeted therapies further complicates treatment of patients. The treatment of TNBC emphasizes enhancing health care and developing personalized medicine. To respond to this need, the researchers have turned their attention to a different approach to scientific enquiry: the era of "big biology" and the integrative study of biological systems, also called "Omics" technologies. The term omics comprises different fields of molecular studies and characterizes a global view on biological molecules such as DNA, RNA, proteins, and metabolites. Combined "omics" approach offers a major tool for the understanding of a challenging cancer model, TNBC. This review discusses the different discoveries made using omics technologies concerning the molecular mechanisms underlying TNBC phenotypic heterogeneity, and their potential transfer to clinical applications.

H3K4 acetylation, H3K9 acetylation and H3K27 methylation in breast tumor molecular subtypes.

AIM: Here, we investigated how the St Gallen breast molecular subtypes displayed distinct histone H3 profiles. PATIENTS & METHODS: 192 breast tumors divided into five St Gallen molecular subtypes (luminal A, luminal B HER2-, luminal B HER2+, HER2+ and basal-like) were evaluated for their histone H3 modifications on gene promoters. RESULTS: ANOVA analysis allowed to identify specific H3 signatures according to three groups of genes: hormonal receptor genes (ERS1, ERS2, PGR), genes modifying histones (EZH2, P300, SRC3) and tumor suppressor gene (BRCA1). A similar profile inside high-risk cancers (luminal B [HER2+], HER2+ and basal-like) compared with low-risk cancers including luminal A and luminal B (HER2-) were demonstrated. CONCLUSION: The H3 modifications might contribute to clarify the differences between breast cancer subtypes.

Epigenetic Modifications with DZNep, NaBu and SAHA in Luminal and Mesenchymal-like Breast Cancer Subtype Cells.

BACKGROUND/AIM: Numerous studies have shown that breast cancer and epigenetic mechanisms have a very powerful interactive relation. The MCF7 cell line, representative of luminal subtype and the MDA-MB 231 cell line representative of mesenchymal-like subtype were treated respectively with a Histone Methyl Transferase Inhibitors (HMTi), 3-Deazaneplanocin hydrochloride (DZNep), two histone deacetylase inhibitors (HDACi), sodium butyrate (NaBu), and suberoylanilide hydroxamic acid (SAHA) for 48 h. MATERIALS AND METHODS: Chromatin immunoprecipitation (ChIP) was used to observe HDACis (SAHA and NaBu) and HMTi (DZNep) impact on histones and more specifically on H3K27me3, H3K9ac and H3K4ac marks with Q-PCR analysis of BRCA1, SRC3 and P300 genes. Furthermore, the HDACi and HMTi effects on mRNA and protein expression of BRCA1, SRC3 and P300 genes were checked. In addition, statistical analyses were used. RESULTS: In the MCF7 luminal subtype with positive ER, H3k4ac was significantly increased on BRCA1 with SAHA. On the contrary, in the MDA-MB 231 breast cancer cell line, representative of mesenchymal-like subtype with negative estrogen receptor, HDACis had no effect. Also, DZNEP decreased significantly H3K27me3 on BRCA1 in MDA-MB 231. Besides, on SRC3, a significant increase for H3K4ac was obtained in MCF7 treated with SAHA. And DZNEP had no effect in MCF7. Also, in MDA-MB 231 treated with DZNEP, H3K27me3 significantly decreased on SRC3 while H3K4ac was significantly increased in MDA-MB-231 treated with SAHA or NaBu for P300. CONCLUSION: Luminal and mesenchymal-like breast cancer subtype cell lines seemed to act differently to HDACis (SAHA and NaBu) or HMTi (DZNEP) treatments.

A bivalent role of TIP60 histone acetyl transferase in human cancer.

Acetylation is a major modification that is required for gene regulation, genome maintenance and metabolism. A dysfunctional acetylation plays an important role in several diseases, including cancer. A group of enzymes-lysine acetyltransferases are responsible for this modification and act in regulation of transcription as cofactors and by acetylation of histones and other proteins. Tip60, a member of MYST family, is expressed ubiquitously and is the acetyltransferase catalytic subunit of human NuA4 complex. This HAT has a well-characterized involvement in many processes, such as cellular signaling, DNA damage repair, transcriptional and cellular cycle. Aberrant lysine acetyltransferase functions promote or suppress tumorigenesis in different cancers such as colon, breast and prostate tumors. Therefore, Tip60 might be a potential and important therapeutic target in the cancer treatment; new histone acetyl transferase inhibitors were identified and are more selective inhibitors of Tip60.

The Role of Soy Phytoestrogens on Genetic and Epigenetic Mechanisms of Prostate Cancer.

Soy phytoestrogens are dietary components with considerable effects on reducing the incidence of prostate cancer. Epidemiological studies demonstrated that occurrence of prostate cancer is relatively low in Asia and Southern Europe, a status associated with consuming of soy isoflavones, such as genistein, daidzein, and glycitein. Soy phytoestrogens exert their activity on molecular mechanisms, including cell-cycle control, induction of apoptosis, inhibition of angiogenesis, and metastasis. In addition, they have antioxidant activity and show regulatory effect on the expression of genes involved in DNA damage and repair. Furthermore, the epigenetic regulation of gene expression can be modified by soy phytoestrogens. They show regulatory effects on gene activity by altering DNA methylation and/or histone modification patterns. In this chapter, we discuss the role of soy phytoestrogens on the genetic and epigenetic mechanisms of prostate cancer. We attempt to provide further insight in order to understand the underlying mechanisms of protective effects of soy phytoestrogens in preventing prostate cancer.

Genome-wide DNA methylation modified by soy phytoestrogens: role for epigenetic therapeutics in prostate cancer?

In prostate cancer, DNA methylation is significantly associated with tumor initiation, progression, and metastasis. Previous studies have suggested that soy phytoestrogens might regulate DNA methylation at individual candidate gene loci and that they play a crucial role as potential therapeutic agents for prostate cancer. The purpose of our study was to examine the modulation effects of phytoestrogens on a genome-wide scale in regards to DNA methylation in prostate cancer. Prostate cancer cell lines DU-145 and LNCaP were treated with 40 µM of genistein and 110 µM of daidzein. DNMT inhibitor 5-azacytidine (2 µM) and the methylating agent budesonide (2 µM) were used to compare their demethylation/methylation effects with phytoestrogens. The regulatory effects of phytoestrogens on DNA methylation were analyzed by using a methyl-DNA immunoprecipitation method coupled with Human DNA Methylation Microarrays (MeDIP-chip). We observed that the methylation profiles of 58 genes were altered by genistein and daidzein treatments in DU-145 and LNCaP prostate cancer cells. In addition, the methylation frequencies of the MAD1L1, TRAF7, KDM4B, and hTERT genes were remarkably modified by genistein treatment. Our results suggest that the modulation effects of phytoestrogens on DNA methylation essentially lead to inhibition of cell growth and induction of apoptosis. Genome-wide methylation profiling reported here suggests that epigenetic regulation mechanisms and, by extension, epigenetics-driven novel therapeutic candidates warrant further consideration in future "omics" studies of prostate cancer.

Prostate cancer: The main risk and protective factors-Epigenetic modifications.

With 13 million new cases worldwide every year, prostate cancer is as a very real public health concern. Prostate cancer is common in over-50s men and the sixth-leading cause of cancer-related death in men worldwide. Like all cancers, prostate cancer is multifactorial - there are non-modifiable risk factors like heredity, ethnicity and geographic location, but also modifiable risk factors such as diet. Diet-cancer linkages have risen to prominence in the last few years, with accruing epidemiological data pointing to between-population incidence differentials in numerous cancers. Indeed, there are correlations between fat-rich diet and risk of hormone-dependent cancers like prostate cancer and breast cancer. Diet is a risk factor for prostate cancer, but certain micronutrients in specific diets are considered protective factors against prostate cancer. Examples include tomato lycopene, green tea epigallocatechin gallate, and soy phytoestrogens. These micronutrients are thought to exert cancer-protective effects via anti-oxidant pathways and inhibition of cell proliferation. Here, we focus in on the effects of phytoestrogens, and chiefly genistein and daidzein, which are the best-researched to date. Soy phytoestrogens are nonsteroid molecules whose structural similarity lends them the ability to mimic the effects of 17ß-estradiol. On top of anti-oxidant effects, there is evidence that soy phytoestrogens can modulate the epigenetic modifications found in prostate cancer. We also studied the impact of phytoestrogens on epigenetic modifications in prostate cancer, with special focus on DNA methylation, miRNA-mediated regulation and histone modifications.

Epigenetic mechanisms of breast cancer: an update of the current knowledge.

Epigenetic alterations are heritable changes in gene expression that occur without causing any change in DNA sequence. They are important key factors for cancer development and prognosis. Breast cancer is induced by the accumulation of altered gene regulation. Besides genetic mutations, epigenetics mechanisms have an important role in breast cancer tumorigenesis. Investigations related with aberrant epigenetic regulations in breast cancer focus on initiating molecular mechanisms in cancer development, identification of new biomarkers to predict breast cancer aggressiveness and the potential of epigenetic therapy. In this review, we will summarize the recent knowledge about the role of epigenetic alterations related with DNA methylation and histone modification in breast cancer. In addition, altered regulation of breast cancer specific genes and the potential of epigenetic therapy will be discussed according to epigenetic mechanisms.

Epigenetic modifications in prostate cancer.

Prostate cancer is the most common cancer in men and the second leading cause of cancer deaths in men in France. Apart from the genetic alterations in prostate cancer, epigenetics modifications are involved in the development and progression of this disease. Epigenetic events are the main cause in gene regulation and the three most epigenetic mechanisms studied include DNA methylation, histone modifications and microRNA expression. In this review, we summarized epigenetic mechanisms in prostate cancer. Epigenetic drugs that inhibit DNA methylation, histone methylation and histone acetylation might be able to reactivate silenced gene expression in prostate cancer. However, further understanding of interactions of these enzymes and their effects on transcription regulation in prostate cancer is needed and has become a priority in biomedical research. In this study, we summed up epigenetic changes with emphasis on pharmacologic epigenetic target agents.