Promoter CpG Methylation Status Research Articles

PRMT5 Mediated HIF1α Signaling and Ras-Related Nuclear Protein as Promising Biomarker in Hepatocellular Carcinoma.

Protein arginine N-methyltransferase 5 (PRMT5) has been identified as a potential therapeutic target for various cancer types. However, its role in regulating the hepatocellular carcinoma (HCC) transcriptome remains poorly understood. In this study, publicly available databases were employed to investigate PRMT5 expression, its correlation with overall survival, targeted pathways, and genes of interest in HCC. Additionally, we utilized in-house generated NGS data to explore PRMT5 expression in dysplastic nodules compared to hepatocellular carcinoma. Our findings revealed that PRMT5 is significantly overexpressed in HCC compared to normal liver, and elevated expression correlates with poor overall survival. To gain insights into the mechanism driving PRMT5 overexpression in HCC, we analyzed promoter CpG islands and methylation status in HCC compared to normal tissues. Pathway analysis of PRMT5 knockdown in the HCC cells revealed a connection between PRMT5 expression and genes related to the HIF1α pathway. Additionally, by filtering PRMT5-correlated genes within the HIF1α pathway and selecting up/downregulated genes in HCC patients, we identified Ras-related nuclear protein (RAN) as a target associated with overall survival. For the first time, we report that PRMT5 is implicated in the regulation of HIF1A and RAN genes, suggesting the potential prognostic utility of PRMT5 in HCC.

Triterpenoid ursolic acid regulates the environmental carcinogen benzo[a]pyrene-driven epigenetic and metabolic alterations in SKH-1 hairless mice for skin cancer interception.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens accountable to developing skin cancers. Recently, we reported that exposure to benzo[a]pyrene (B[a]P), a common PAH, causes epigenetic and metabolic alterations in the initiation, promotion and progression of non-melanoma skin cancer (NMSC). As a follow-up investigation, this study examines how dietary triterpenoid ursolic acid (UA) regulates B[a]P-driven epigenetic and metabolic pathways in SKH-1 hairless mice. Our results show UA intercepts against B[a]P-induced tumorigenesis at different stages of NMSC. Epigenomic cytosines followed by guanine residues (CpG) methyl-seq data showed UA diminished B[a]P-mediated differentially methylated regions (DMRs) profiles. Transcriptomic RNA-seq revealed UA revoked B[a]P-induced differentially expressed genes (DEGs) of skin cancer-related genes, such as leucine-rich repeat LGI family member 2 (Lgi2) and kallikrein-related peptidase 13 (Klk13), indicating UA plays a vital role in B[a]P-mediated gene regulation and its potential consequences in NMSC interception. Association analysis of DEGs and DMRs found that the mRNA expression of KLK13 gene was correlated with the promoter CpG methylation status in the early-stage comparison group, indicating UA could regulate the KLK13 by modulating its promoter methylation at an early stage of NMSC. The metabolomic study showed UA alters B[a]P-regulated cancer-associated metabolisms like thiamin metabolism, ascorbate and aldarate metabolism during the initiation phase; pyruvate, citrate and thiamin metabolism during the promotion phase; and beta-alanine and pathothenate coenzyme A (CoA) biosynthesis during the late progression phase. Taken together, UA reverses B[a]P-driven epigenetic, transcriptomic and metabolic reprogramming, potentially contributing to the overall cancer interception against B[a]P-mediated NMSC.

The environmental carcinogen benzo[a]pyrene regulates epigenetic reprogramming and metabolic rewiring in a two-stage mouse skin carcinogenesis model.

Non-melanoma skin cancer (NMSC) is the most common cancer in the world. Environmental exposure to carcinogens is one of the major causes of NMSC initiation and progression. In the current study, we utilized a two-stage skin carcinogenesis mouse model generated by sequential exposure to cancer-initiating agent benzo[a]pyrene (BaP) and promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA), to study epigenetic, transcriptomic and metabolic changes at different stages during the development of NMSC. BaP/TPA caused significant alterations in DNA methylation and gene expression profiles in skin carcinogenesis, as evidenced by DNA-seq and RNA-seq analysis. Correlation analysis between differentially expressed genes and differentially methylated regions found that the mRNA expression of oncogenes leucine rich repeat LGI family member 2 (Lgi2), kallikrein-related peptidase 13 (Klk13) and SRY-Box transcription factor (Sox5) are correlated with the promoter CpG methylation status, indicating BaP/TPA regulates these oncogenes through regulating their promoter methylation at different stages of NMSC. Pathway analysis identified that the modulation of macrophage-stimulating protein-recepteur d'origine nantais and high-mobility group box 1 signaling pathways, superpathway of melatonin degradation, melatonin degradation 1, sirtuin signaling and actin cytoskeleton signaling pathways are associated with the development of NMSC. The metabolomic study showed BaP/TPA regulated cancer-associated metabolisms like pyrimidine and amino acid metabolisms/metabolites and epigenetic-associated metabolites, such as S-adenosylmethionine, methionine and 5-methylcytosine, indicating a critical role in carcinogen-mediated metabolic reprogramming and its consequences on cancer development. Altogether, this study provides novel insights integrating methylomic, transcriptomic and metabolic-signaling pathways that could benefit future skin cancer treatment and interception studies.

Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas.

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another case. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. Predominant type-A EBV variant and LMP1 30-bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer.

PTEN-knockout regulates metabolic rewiring and epigenetic reprogramming in prostate cancer and chemoprevention by triterpenoid ursolic acid.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is one of the most frequently mutated/deleted tumor suppressor genes in many human cancers. Ursolic acid (UA) is a natural triterpenoid possessing antioxidant, anti-inflammatory, and anticancer effects. However, how PTEN impacts metabolic rewiring and how UA modifies PTEN-driven metabolic and epigenetic reprogramming in prostate cancer (PCa) remains unknown. In the current study, we found that UA protects against PTEN knockout (KO)-induced tumorigenesis at different stages of PCa. Epigenomic CpG methyl-seq revealed UA attenuated PTEN KO-induced differentially methylated regions (DMRs) profiles. Transcriptomic RNA-seq showed UA abrogated PTEN KO-induced differentially expressed genes (DEGs) of PCa-related oncogenes' Has3, Cfh, and Msx1 overexpression, indicating UA plays a crucial role in PTEN KO-mediated gene regulation and its potential consequences on cancer interception. Association analysis of DEGs and DMRs identified that the mRNA expression of tumor suppressor gene BDH2, and oncogenes Ephas, Isg15, and Nos2 were correlated with the promoter CpG methylation status in the early-stage comparison groups indicating UA could regulate the oncogenes or tumor suppressor genes by modulating their promoter methylation at an early stage of prostate tumorigenesis. The metabolomic study showed UA attenuated PTEN KO-regulated cancer-associated metabolisms like purine metabolism/metabolites correlating with RNAseq findings, glycolysis/gluconeogenesis metabolism, as well as epigenetic-related metabolites pyruvate and lactate indicating UA plays a critical role in PTEN KO-mediated metabolic and epigenetic reprogramming and its consequences on cancer development. In this context, UA impacts metabolic rewiring causing epigenetic and transcriptomic reprogramming potentially contributing to the overall protection against prostate-specific PTEN KO-mediated PCa.

Rlip Depletion Alters Oncogene Transcription at Multiple Distinct Regulatory Levels.

Rlip76 (Rlip) is a multifunctional membrane protein that facilitates the high metabolic rates of cancer cells through the efflux of toxic metabolites and other functions. Rlip inhibition or depletion results in broad-spectrum anti-cancer effects in vitro and in vivo. Rlip depletion effectively suppresses malignancy and causes global reversion of characteristic CpG island methylomic and transcriptomic aberrations in the p53-null mouse model of spontaneous carcinogenesis through incompletely defined signaling and transcriptomic mechanisms. The methylome and transcriptome are normally regulated by the concerted actions of several mechanisms that include chromatin remodeling, promoter methylation, transcription factor interactions, and miRNAs. The present studies investigated the interaction of Rlip depletion or inhibition with the promoter methylation and transcription of selected cancer-related genes identified as being affected by Rlip depletion in our previous studies. We constructed novel promoter CpG island/luciferase reporter plasmids that respond only to CpG methylation and transcription factors. We found that Rlip depletion regulated expression by a transcription factor-based mechanism that functioned independently of promoter CpG methylation, lipid peroxidation, and p53 status.

Nfe2l2 Regulates Metabolic Rewiring and Epigenetic Reprogramming in Mediating Cancer Protective Effect by Fucoxanthin.

Fucoxanthin (FX) is a carotenoid with many pharmaceutical properties due to its antioxidant/anti-inflammatory and epigenetic effects. NFE2L2 is involved in the defense against oxidative stress/inflammation-mediated diseases, like anticancer effects elicited by phytochemicals including FX. However, the role of FX and NFE2L2 in metabolic rewiring, epigenomic reprogramming, and transcriptomic network in blocking pro-tumorigenic signaling and eliciting cancer-protective effects remains unknown. Herein, we utilized multi-omics approaches to evaluate the role of NFE2L2 and the impact of FX on tumor promoter TPA-induced skin cell transformation. FX blocked TPA-induced ROS and oxidized GSSG/reduced GSH in Nfe2l2wild-type(WT) but not Nfe2l2-knockdown (KD) cells. Both Nfe2l2 KD and TPA altered cellular metabolisms and metabolites which are tightly coupled to epigenetic machinery. The suppressive effects of FX on TPA-enhancedSAM/SAH was abrogated by Nfe2l2 KD indicating Nfe2l2 plays a critical role in FX-mediated metabolic rewiring and its potential consequences on epigenetic reprogramming. Epigenomic CpG methyl-seq revealed that FX attenuated TPA-induced differentially methylated regions (DMRs) of Uhrf1 and Dnmt1 genes. Transcriptomic RNA-seq showed that FX abrogated TPA-induced differentially expressed genes (DEGs) of Nfe2l2-related genes Nqo1, Ho1, and Keap1. Associative analysis of DEGs and DMRs identified that the mRNA expressions of Uhrf1 and Dnmt1 were correlated with the promoter CpG methylation status. Chromatin immunoprecipitation assay showed that FX restored Uhrf1 expression by regulating H3K27Me3 enrichment in the promoter region. In this context, FX/Nfe2l2's redox signaling drives metabolic rewiring causing epigenetic and transcriptomic reprogramming potentially contributing to the protection of TPA-induced JB6 cellular transformation skin cancer model. Graphical abstract.

Triterpenoid ursolic acid drives metabolic rewiring and epigenetic reprogramming in treatment/prevention of human prostate cancer.

Ursolic acid (UA) is a triterpenoid phytochemical with a strong anticancer effect. The metabolic rewiring, epigenetic reprogramming, and chemopreventive effect of UA in prostate cancer (PCa) remain unknown. Herein, we investigated the efficacy of UA in PCa xenograft, and its biological effects on cellular metabolism, DNA methylation, and transcriptomic using multi-omics approaches. The metabolomics was quantified by liquid-chromatography-mass spectrometry (LC-MS) while epigenomic CpG methylation in parallel with transcriptomic gene expression was studied by next-generation sequencing technologies. UA administration attenuated the growth of transplanted human VCaP-Luc cells in immunodeficient mice. UA regulated several cellular metabolites and metabolism-related signaling pathways including S-adenosylmethionine (SAM), methionine, glucose 6-phosphate, CDP-choline, phosphatidylcholine biosynthesis, glycolysis, and nucleotide sugars metabolism. RNA-seq analyses revealed UA regulated several signaling pathways, including CXCR4 signaling, cancer metastasis signaling, and NRF2-mediated oxidative stress response. Epigenetic reprogramming study with DNA Methyl-seq uncovered a list of differentially methylated regions (DMRs) associated with UA treatment. Transcriptome-DNA methylome correlative analysis uncovered a list of genes, of which changes in gene expression correlated with the promoter CpG methylation status. Altogether, our results suggest that UA regulates metabolic rewiring of metabolism including SAM potentially driving epigenetic CpG methylation reprogramming, and transcriptomic signaling resulting in the overall anticancer chemopreventive effect.

Promoter CpG methylation status affects ADRP gene expression level and intramuscular fat content in pigs

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed early during adipose differentiation. However, the underlying mechanism is still unclear. In this study, the porcine ADRP (pADRP) gene regulatory mechanism and its relationship to intramuscular fat (IMF) content are analysed. We investigated the characteristics of the pADRP gene prompter as well as its transcription level, core promoter and epigenetic modification status using real-time PCR, Dual-luciferase reporter assays and bisulphite sequencing. The results showed that pADRP mRNA expression in Longissimus Dorsi (LD) muscle of the low IMF (LEAN) group was lower than that in the high IMF (FAT) group (p<.01). Maximum promoter activity was found in the −926 bp to −235 bp region. The methylation level of the pADRP of this region (mainly 7th, 10th, 12th CpG sites) in the low IMF group (86.67%) was significantly higher than that in the high IMF group (46.7%) (p<.01). Our result plays an important role in pADRP transcriptional regulation and provides a good prospect for the marbling traits improvement. Highlights pADRP expression differs in divergent IMF content pigs. Promoter region located −929 to −235 bp, and its methylation contributes to gene expression.

BTB/POZ zinc finger protein ZBTB16 inhibits breast cancer proliferation and metastasis through upregulating ZBTB28 and antagonizing BCL6/ZBTB27

BackgroundBreast cancer remains in urgent need of reliable diagnostic and prognostic markers. Zinc finger and BTB/POZ domain-containing family proteins (ZBTBs) are important transcription factors functioning as oncogenes or tumor suppressors. The role and regulation of ZBTB16 in breast cancer remain to be established.MethodsReverse-transcription PCR and methylation-specific PCR were applied to detect expression and methylation of ZBTB16 in breast cancer cell lines and tissues. The effects of ZBTB16 in breast cancer cells were examined via cell viability, CCK8, Transwell, colony formation, and flow cytometric assays. Xenografts and immunohistochemistry analyses were conducted to determine the effects of ZBTB16 on tumorigenesis in vivo. The specific mechanisms of ZBTB16 were further investigated using Western blot, qRT-PCR, luciferase assay, and co-IP.ResultsZBTB16 was frequently downregulated in breast cancer cell lines in correlation with its promoter CpG methylation status. Restoration of ZBTB16 expression led to induction of G2/M phase arrest and apoptosis, inhibition of migration and invasion, reversal of EMT, and suppression of cell proliferation, both in vitro and in vivo. Furthermore, ectopically expressed ZBTB16 formed heterodimers with ZBTB28 or BCL6/ZBTB27 and exerted tumor suppressor effects through upregulation of ZBTB28 and antagonistic activity on BCL6.ConclusionsLow expression of ZBTB16 is associated with its promoter hypermethylation and restoration of ZBTB16 inhibits tumorigenesis. ZBTB16 functions as a tumor suppressor through upregulating ZBTB28 and antagonizing BCL6. Our findings also support the possibility of ZBTB16 being a prognostic biomarker for breast cancer.

Epigenome, Transcriptome, and Protection by Sulforaphane at Different Stages of UVB-Induced Skin Carcinogenesis.

Sulforaphane (SFN), a potent antioxidant and antiinflammatory agent, has been shown to protect against cancers especially at early stages. However, how SFN affects UVB-mediated epigenome/DNA methylome and transcriptome changes in skin photodamage has not been fully assessed. Herein, we investigated the transcriptomic and DNA methylomic changes during tumor initiation, promotion, and progression and its impact and reversal by SFN using next-generation sequencing (NGS) technology. The results show that SFN reduced tumor incidence and tumor number. SFN's protective effects were more dramatic in the early stages than with later stages. Bioinformatic analysis of RNA sequencing (RNA-seq) data shows differential expressed genes and identifies the top canonical pathways related to SFN treatment of UVB-induced different stages of epidermal carcinogenesis. These pathways include p53 signaling, cell cycle: G2-M DNA damage checkpoint regulation, Th1, and Th2 activation pathway, and PTEN signaling pathways. The top upstream regulators related to UVB and SFN treatment as time progressed include dextran sulfate, TP53, NFE2L2 (Nrf2), IFNB1, and IL10RA. Bioinformatic analysis of Methyl-seq data shows several differential methylation regions induced by UVB were attenuated by SFN. These include Notch1, Smad6, Gnai3, and Apc2 Integrative analysis of RNA-seq and DNA-seq/CpG methylome yields a subgroup of genes associated with ultraviolet B (UVB) and SFN treatment. The changes in gene expression were inversely correlated with promoter CpG methylation status. These genes include Pik3cd, Matk, and Adm2 In conclusion, our study provides novel insights on the impact of SFN on the transcriptomic and DNA methylomic of UVB-induced different stages of skin cancer in mice.

527 Promoter CpG Methylation Status in POMC and its Role in Dyspigmented Burn Hypertrophic Scar

527 Promoter CpG Methylation Status in POMC and its Role in Dyspigmented Burn Hypertrophic Scar

Tumor suppressive BTB/POZ zinc-finger protein ZBTB28 inhibits oncogenic BCL6/ZBTB27 signaling to maintain p53 transcription in multiple carcinogenesis.

Zinc-finger and BTB/POZ domain-containing family proteins (ZBTB) are important transcription factors functioning as tumor suppressors or oncogenes, such as BCL6/ZBTB27 as a key oncoprotein for anti-cancer therapy. Through epigenome study, we identified ZBTB28/BCL6B/BAZF, a BTB/POZ domain protein highly homologous to BCL6, as a methylated target in multiple tumors. However, the functions and mechanism of ZBTB28 in carcinogenesis remain unclear. Methods: ZBTB28 expression and methylation were examined by reverse-transcription PCR and methylation-specific PCR. The effects and mechanisms of ectopic ZBTB28 expression on tumor cells were assessed with molecular biological and cellular approaches in vitro and in vivo. Results: Albeit broadly expressed in multiple normal tissues, ZBTB28 is frequently downregulated in aero- and digestive carcinoma cell lines and primary tumors, and correlated with its promoter CpG methylation status. Further gain-of-function study showed that ZBTB28 functions as a tumor suppressor inhibiting carcinoma cell growth in vitro and in vivo, through inducing cell cycle arrest and apoptosis of tumor cells. ZBTB28 suppresses cell migration and invasion by reversing EMT and cell stemness. ZBTB28 transactivates TP53 expression, through binding to the p53 promoter in competition with BCL6, while BCL6 itself was also found to be a direct target repressed by ZBTB28. Conclusion: Our results demonstrate that ZBTB28 functions as a tumor suppressor through competing with BCL6 for targeting p53 regulation. This newly identified ZBTB28/BCL6/p53 regulatory axis provides further molecular insight into carcinogenesis mechanisms and has implications in further improving BCL6-based anticancer therapy.

Promoter Hypermethylation Analysis of the Tumor Suppressor Genes RASSF1A and RASSF2A in Iranian Endometrial Carcinoma Patients

Introduction: The endometrial cancer (EC) is the seventh most common malignancies worldwide among females with good prognosis in early stages of the disease. The CpG Island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we investigated the methylation pattern in promoter region of RASSF1A and RASSF2A genes in Endometrial cancer patients in Iranian women to identify correlations among promoter hypermethylation, disease risk and clinicopathological parameters. Methods: 28 patients and 22 healthy controls were studied. Isolation of genomic DNA from FFPE and peripheral blood was performed and Methylation-Specific PCR (MSP) was applied for analysis of the promoter CpG methylation status of RASSF1A and RASSF2A genes in the studied population. Results: A significant difference was found among the study groups and the presence of promoter CpG hypermethylation status in the RASSF1A (P = 0.0321) and RASSF2A (P = 0.0003) genes. RASSF1A, and RASSF2A gene promoter methylations were present in 53.57% and 42.85% of EC samples when compared to those in the controls with 31.81% and 9.09% respectively. Furthermore, methylation status between tissue and blood samples of RASSF1A, and RASSF2A genes was not significant (P = 0.49 and 0.09 respectively). Our results indicated a corollation between ages, menosososal state and tumor grade with RASSF1A, and RASSF2A promoter methylation. Conclusions: In our study, Hypermethylation of both RASSF1A and RASSF2A genes are important events in carcinogenesis of endometrial cancer. Epigenetic alternations may have diagnostic value for early diagnosis and better clicinal management of susceptibility to endometrial malignancies.

Searching potential transcription factors regulating MMP13 overexpression via promoter CpG methylation status in human osteoarthritic chondrocytes: A microarray screening

Purpose: Matrix metalloproteinase 13 (MMP-13) is known to play important roles in the progress of osteoarthritis (OA) in human articular cartilage. Our previous studies have demonstrated that the elevated MMP13 gene expression in human chondrocytes is regulated by the CpG methylation status in its proximal promoter. Also, hypoxia-inducible factor (HIF) -2α, a transcription factor (TF) mainly induced under the hypoxic condition, was found to regulate MMP13 expression via methylation status of a specific CpG site in the proximal promoter. In this study, we screened TFs that have a potential to regulate MMP13 expression via its promoter CpG methylation status by a microarray analysis comparing healthy and OA human chondrocytes. Methods: Four healthy and five OA cartilage pieces were harvested from the human femoral head collected from the hemiarthroplasty for fracture of neck of femur (#NOF) and total hip arthroplasty for hip OA, respectively. Then mRNA was extracted (QIAGEN, RNeasy Lipid Kit®), from those cartilage pieces. The mRNA samples with RNA integrity number (RIN) >6.5 by Bioanalyzer® (Agilent Technologies) were proceeded to a microarray analysis (TORAY, 3D-Gene®). The expression level of MMP13 and various TF genes were compared between healthy and OA human chondrocytes. The TF gene expression with 2-time difference between healthy and OA chondrocytes were selected for further analysis. The potential binding sites of the selected TFs, and the presence of CpG sites in those binding sites, were detected in up to −5000bp of MMP13 promoter using TF binding site databases. Results: The relative expression levels of MMP13 were 32 ± 43 and 538 ± 337 in healthy and OA chondrocytes, respectively (P=0.022). In the microarray analysis, 41 TFs were more expressed, and 40 TFs were less expressed in OA compared to healthy chondrocytes, respectively. Of them, 13 TFs were found to be potentially bindable to up to −5000-bp of MMP13 promoter. Moreover, transcription factors FOXO1A, NFATC2 and 4, FOXM1, FOS, LEF1 and E2F2 were found to have their consensus sequences close to CpG sites in the MMP13 promoter sequence. Conclusions: In addition to HIF-2α, the function of FOXO1A, NFATC2 and 4, FOXM1, FOS, LEF1 or E2F2 can be also regulated by the methylation status of CpG sites in MMP13 promoter. Further investigation is needed to prove the detailed function of those transcription factors, especially for a relationship with CpG methylation status of the MMP13 promoter.

Probable Chemical Hypoxia Effects on Progress of CNV Through Induction of Promoter CpG Demethylation and Overexpression of IL17RC in Human RPE Cells

ABSTRACTPurpose: To survey the changes of promoter CpG methylation status and mRNA expression of IL17RC (interleukin 17 receptor C) gene in retinal pigment epithelium (RPE) cells under chemical hypoxia condition for choroidal neovascularization (CNV) modeling in vitro.Materials and Methods: RPE cells were cultured in both untreated as a control group and treated by cobalt chloride media as a hypoxia group for various concentrations (100–150μM) and times (24–36 hrs.) To confirm chemical hypoxia condition, mRNA expression of HIF (Hypoxia Inducible Factor) -1α, -2α, and Vascular Endothelial Growth Factor (VEGF) was compared between two groups by Real-time PCR. Also, in normoxia and hypoxia conditions, IL17RC expression changes and promoter CpG methylation status were evaluated by Real-time PCR and methylation-specific PCR (MSP) techniques, respectively.Results: Overexpression of HIF-1α, HIF-2α, and VEGF was significant in hypoxia versus normoxia conditions. Our data showed overexpression of IL17RC (2.1- to 6.3-fold) and decreasing of its promoter methylation in comparison with hypoxia and normoxia conditions. It was found that there are significant association between promoter methylation status and expression of IL17RC in chemical hypoxia condition.Conclusion: Therefore, methylation of IL17RC could play as a marker in CNV and degeneration of RPE cells in vitro. Additionally, HIF-α and methylation phenomena may be considered as critical targets for blocking in angiogenesis of age-related degeneration in future studies.

Modulation of p21, DAPK1 and COX-2 during the DMBA/TPA-induced mouse skin tumorigenesis and its prevention by phytic acid

Chemoprevention by naturally occurring agents is gaining much attention as a newer dimension in the management of cancer. Many naturally occurring agents have shown cancer chemopreventive potential in a variety of bioassay systems and animal models, having relevance to human disease. Phytic acid or Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of Phytic acid against the 7, 12-dimethylbenz [a] anthracene (DMBA)/ 12-O-tetradecanoylphorbol-13- acetate (TPA) induced mouse skin tumorigenesis at 4 and 16 weeks, the time before and after the tumor development. At molecular level we studied expression and promoter CpG methylation status of p21, DAPK1 and COX-2. Our data suggests exposure of DMBA/TPA methylated the promoter region of p21 and DAPK1 genes in time dependent manner that could be the cause of down regulation of their expression with time, which were reversed by administration of phytic acid. But we did not observe methylation in COX-2 whereas upregulation of COX-2 was observed at protein level in mice treated with DMBA followed by TPA in time dependent manner. Administration of phytic acid prevented theses DMBA/TPA induced molecular changes. Study provides a rationale for cancer chemoprevention by natural occurring compounds like Phytic acid.

Collagen Triple Helix Repeat Containing 1 (CTHRC1) acts via ERK-dependent induction of MMP9 to promote invasion of colorectal cancer cells

Collagen triple helix repeat-containing 1 (CTHRC1) is known to be aberrantly upregulated in most human solid tumors, although the functional roles of CTHRC1 in colorectal cancer remain unclear. In this study, we investigated the occurrence of CTHRC1 upregulation and its role in vivo and in vitro. The expression profile and clinical importance of CTHRC1 were examined by reverse transcription-polymerase chain reaction and immunohistochemical analyses in normal and tumor patient samples. CTHRC1 was detectable in normal tissues, but also was highly expressed in tumor specimens. CTHRC1 upregulation was significantly associated with demethylation of the CTHRC1 promoter in colon cancer cell lines and tumor tissues. Clinicopathologic analyses showed that nodal status and expression of CTHRC1 (95% CI 0.999-3.984, p=0.05) were significant prognostic factors for disease-free survival. Promoter CpG methylation and hypermethylation status were measured by bisulfite sequencing and pyrosequencing analysis. Furthermore, we showed that overexpression of CTHRC1 in the SW480 and HT-29 cell lines increased invasiveness, an effect mediated by extracellular signal-regulated kinase (ERK)-dependent upregulation of matrix metalloproteinase 9 (MMP9). Consistent with this, we found that knockdown of CTHRC1 attenuated ERK activation and cancer cell invasivity. These results demonstrate that CTHRC1 expression is elevated in human colon cancer cell lines and clinical specimens, and promotes cancer cell invasivity through ERK-dependent induction of MMP9 expression. Our results further suggest that high levels of CTHRC1 expression are associated with poor clinical outcomes.

Promoter hypermethylation of the tumor-suppressor genes RASSF1A, GSTP1 and CDH1 in endometrial cancer

Endometrial cancer is a common gynecological malignancy with a good prognosis in early stages of the disease. The CpG island in the promoter region of tumor-suppressor genes are frequently methylated in various types of human cancers. In the present study, we examined the methylation status of the GSTP1, CDH1 and RASSF1A genes in endometrioid endometrial cancer (EEC), endometrial complex hyperplasia (EHP) and healthy endometrium with the aim to identify correlations between promoter hypermethylation, disease risk and clinicopathological parameters. A nested two-stage methylation-specific PCR (MSP) was performed to analyze the promoter CpG methylation status of GSTP1, CDH1 and RASSF1A genes in the population studied. A total of 92 subjects were initially included in the study of which 41 EEC, 19 EHP and 20 controls were processed for final analyses. A significant difference was found between the study groups and the presence of promoter CpG hypermethylation status in the GSTP1 (p<0.05) and RASSF1A (p<0.0001) genes. RASSF1A, GSTP1 and CDH1 gene promoter methylation was present in 85.4, 68.3 and 31.4% of EEC samples when compared to that in the controls with 30.0, 35.0 and 20.0%, respectively. CpG methylation of all three investigated tumor-suppressor genes was found in 12.2% of EEC patients, in 4.2% of EHP patients and in 3.7% of the controls, respectively. Positive findings for the promoter methylation of two investigated genes were found in 48.7% of EEC patients, 26.0% of EHP patients and in 18.5% of the controls. With regard to histopathological variables and CpG methylation, we found significant correlations between the RASSF1A and GSTP1 genes and higher tumor grade, deeper myometrial invasion and positive metastatic involvement of pelvic lymph nodes. No associations were noted between promoter hypermethylation of the CDH1 gene and biological features of the endometrial cancer cases. The results indicate that aberrant CpG methylation of the promoter region in the GSTP1 and RASSF1A tumor-suppressor genes is an important event in carcinogenesis of endometrial cancer and may have an impact on tumor aggressiveness. Finally, the present study suggests that epigenetic alterations may be of diagnostic value for the better clinical management of premalignant endometrial lesions.

Usefulness of Peripheral Blood Circulating DNAs As an Alternative to DNA From Bone Marrow Cells to Detect CpG Global Methylation Status and Genetic Mutations in Patients with Myelodysplastic Syndromes

Abstract 3854 Background:Several genetic/epigenetic abnormalities are associated with the pathogenesis of myelodysplastic syndromes (MDS). DNA methyltransferase inhibitors (DNMTi), azacitidine and decitabine, have recently come to be considered as standard therapeutics for patients with MDS. However, biomarkers that predict the effectiveness of DNMTi before and/or during treatment are still lacking. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic status, repeated sampling is difficult because of pain and safety concerns. Therefore, alternatives are required. One possibility is to use circulating cell-free DNA in the plasma and serum of peripheral blood (PB); a technique previously used for the detection of genetic/epigenetic abnormalities in solid tumor cells. Aims:Assess the quality of PB circulating DNA from patients with MDS, and investigate its usefulness for analyzing serial genetic/epigenetic changes following treatment. Methods:DNA from BM, PB mononuclear cells (MNC), plasma and serum were obtained repeatedly from MDS patients and visualized via agarose gel electrophoresis. Bisulfite conversion of genomic or circulating DNA was performed and then pyrosequencing performed for the 4 CpG sites of long interspersed nuclear elements-1 (LINE-1) elements to detect global methylation. Bisulfite pyrosequencing for the 5 CpG sites of p15INK4B gene promoter was also performed. Genetic mutations were screened firstly by single nuclear polymorphism (SNP) array analysis and confirmed by DNA sequencing for the specific gene mutations. BM cells were sorted into CD34(+)/CD38(-), CD34(+)/CD38(+), and CD34(-) subpopulations, and the percentage of mutated DNA confirmed by pyrosequencing analysis. Results and Discussion:Circulating DNA from both healthy volunteer donors and MDS patients showed a ladder pattern of DNA fragments 160∼180 base pairs apart, suggesting their accumulation from mono-/di-nucleosomes. The plasma DNA concentration was relatively higher in patients with higher BM blast cell counts. Plasma DNA concentration had been changed even in the same patients according to the disease status. CpG methylation status of the LINE-1 promoter after treatment with azacitidine (days 1 to 28) was analyzed by pyrosequencing. Although the methylation status of 1 patient did not show any significant change, other 2 patients showed a serial decrease of the methylation percentage confirmed at days 3, 6, and 9 using DNA from plasma, serum, and PBMNC. Plasma DNA showed more rapid and significant changes at days 3 (p<0.001) and 6 (p<0.05) compared with serum DNA (not significant (N.S.) at day 3 and 6) and PBMNC (N.S. at day 3 and p<0.05 at day 6). These data suggest that circulating DNA from plasma can be used for analysis of the LINE-1 promoter as a measure of global methylation as an alternative strategy to using MNC in PB and/or BM. The changing ratio is different among patients and it may reflect the efficacy of DNMTi or the amount of MDS clones. p15INK4B gene promoter CpG methylation status was analyzed using BM cells, PBMNC, and circulating DNA. The methylation percentage was not significantly different among whole BM cells and 3 BM subpopulations, and plasma and serum DNA showed similar methylation pattern as whole BM DNA. Next, PB circulating DNA was utilized for the detection of genetic mutations in MDS cells. TET2 mutation (Y1245Term; TAC to TAG) was confirmed in a patient showing chromosome 4q uni-parental disomy (UPD), and the existence ratio of the mutation was significantly higher in plasma and serum DNA than in CD34(-) BM cells (p<0.05 and p<0.01, respectively) and almost equivalent to that in the CD34(+)/38(-) BM stem cell population. The high percentage of mutated genes in circulating DNA may result from abnormal DNA being released from fragile MDS clones, enriching the circulating DNA compared with DNA from BM or PBMNC but further molecular analyses and higher patient numbers are required to confirm this. Conclusions:PB circulating DNA can be reliably and sensitively used to detect epigenetic changes on genomic DNA after treatment with DNMTi. Genetic mutations in MDS clones can also be detected sensitively, at a level almost equivalent to that in BM CD34+/38- stem cells. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a better, less painful and safer alternative to using BM aspiration. Disclosures:No relevant conflicts of interest to declare.