Demethylating Agent Research Articles

Abstract 2660: A renal CpG island methylator phenotype (R-CIMP) in kidney tumors associated with germline mutations of FH and SDHB

Abstract Germline mutations of the genes encoding the Krebs cycle enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDHB, SDHC, SDHD) have both been associated with an increased risk of renal cell carcinoma (RCC). These renal tumors demonstrate complete loss of enzyme activity and significantly increased levels of intracellular fumarate or succinate that are predicted to infer with the actions of multiple cellular processes primarily through the inhibition of 2-oxoglutarate-dependent dioxygenases, such as the prolyl hydroxylases that regulate the HIF pathway and the TET enzymes that regulate DNA methylation. This study evaluated and compared the genome-wide methylation profiles of 33 tumor samples (including 9 FH -/- and 7 SDHB -/- tumors), 10 associated normal samples and 13 cell-lines (including cell-line models derived from FH -/- and SDHB -/- tumors) using the Illumina HumanMethylation450 BeadChip arrays. Tumors derived from patients with germline FH or SDHB mutations demonstrated a distinct pattern of hypermethylation across the genome with enrichment for hypermethylation within the CpG island regions. SDHB mutation associated tumors demonstrated that was more variable and less extensive pattern of hypermethylation than the tumors associated with germline FH mutations. Cell-line models derived from HLRCC or SDHB tumors demonstrated patterns of hypermethylation similar to the patterns observed in the tumors, yet re-expression of either wild-type FH or SDHB within these cell-line models did not alter the methylation profiles. All the HLRCC and SDHB-RCC cell lines, including the cell-line re-expressing of either wild-type FH or SDHB, demonstrated growth inhibition and reduced invasion rates in response to treatment with 5-aza-2′-deoxycytidine. Thus, renal cell carcinomas associated with FH or SDHB germline mutation demonstrate a specific R-CIMP hypermethylation profile that differentiates these tumors from tumors derived from other RCC susceptibility syndromes. These tumors represent the most aggressive types of familial-associated RCC and this could provide a useful biomarker for identifying these tumors and de-methylating agents, such as 5-aza-2′-deoxycytidine, may be effective as part of a therapeutic regime for the treatment of these tumors. Citation Format: Christopher Ricketts, J. Keith Killian, Cathy D. Vocke, Carole Sourbier, Youfeng Yang, Maria J. Merino, Paul S. Meltzer, W. Marston Linehan. A renal CpG island methylator phenotype (R-CIMP) in kidney tumors associated with germline mutations of FH and SDHB. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2660.

Abstract 4467: PRSS8 methylation and its significance in esophageal squamous cell carcinoma

Abstract Esophageal cancer is one of the most common cancers worldwide, and the incidence and mortality is increasing rapidly in recent years in China, but the underlying mechanisms are largely unclear. Herein we used immunohistochemical staining on an esophageal squamous cell carcinoma (ESCC) tissue microarray (TMA) containing 362 cases of ESCC and found that the expression of PRSS8, serine protease prostasin, was significantly decreased in ESCC. Interestingly, the reduction of PRSS8 was well correlated with poor differentiation, less stromal lymphocyte infiltration, and shorter survival time. PRSS8 mRNA levels were also significantly reduced in ESCC compared to the adjacent normal esophagus assayed by quantitative real-time PCR. Methylation specific PCR showed that PRSS8 promoter region was hypermethylated in ESCC tissues and cell lines, which was associated with downregulation of PRSS8 expression and decreased activities of PRSS8 promoter. Promoter deletion and truncation approaches showed that the CpG island in PRSS8 promoter region played an important role on the determination of promoter activities. De-methylation agent decitabine was able to restrore PRSS8 expression in esophageal cancer cell lines, leading to the inhibition of cancer cell proliferation, motility, migration and cell cycle arrest. However, the restoration of PRSS8 and its tumor inhibition could be reversed by small interfering RNA targeting PRSS8. Immunoblotting results showed that tumor inhibition of PRSS8 may be associated with proliferation- and epithelial-mesenchymal transition - related proteins (e.g. upregulation of E-cadherin and downregulation of Twist and Snail) in ESCC cells. The findings from current study strongly suggested that PRSS8 acted as a tumor suppressor and PRSS8 promoter region hypermethylation has clinical significance of in ESCC. Citation Format: Yonghua Bao, Qian Wang, Wancai Yang. PRSS8 methylation and its significance in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4467.

Epigenetic silencing of TPM2 contributes to colorectal cancer progression upon RhoA activation.

Beta-tropomyosin (β-tropomyosin, TPM2) has been found to be downregulated in colorectal cancer (CRC) in previous studies. In this study, we aimed to investigate the mechanisms and potential biological consequences of the downregulation of TPM2 in colorectal cancer. TPM2 expression in colorectal cancer was assessed by qRT-PCR and immunostaining. The biological functions of TPM2 were assessed in cell lines either overexpressing or underexpressingTPM2. Aberrant DNA methylation in the promoter region is associated with suppression of TPM2 expression in primary colorectal cancer tissue samples. Treatment with the demethylation agent 5-AZA can induceTPM2 expression in colorectal cancer cell lines. Reconstitution of TPM2 suppresses cell proliferation and migration in colorectal cancer cell lines, whereas the loss of TPM2 expression is associated with increased tumor proliferation and migration in vitro, which was accompanied by RhoA activation. In summary, our findings indicate that TPM2 appears to be commonly silenced by aberrant DNA methylation in colon cancer. TPM2 loss is associated with RhoA activation and tumor proliferation.

Aberrant DNA methylation-induced gene inactivation is associated with the diagnosis and/or therapy of T-cell leukemias

Aberrant hypermethylation of tumor suppressor genes is known to play an important role in the development of many tumors, and aberrant DNA hypermethylation was recently identified in hematologic malignancies, where it is thought to hold relevance in leukemogenesis. Here, we report that there are differences in the DNA methylation patterns seen in normal peripheral blood and two T-cell leukemia cell lines. We identify nine genes (CLEC4E, CR1, DBC1, EPO, HAL-DOA, IGF2, IL12B, ITGA1, and LMX1B) that are significantly hypermethylated in T-cell leukemias cell lines, and suggest that aberrant hypermethylation of these normally unmethylated genes may induce their transcriptional and expressional silencing. Furthermore, we observed that the expression levels of DNMT1 and DNMT3a were significantly decreased by 5-aza-2′-deoxycytidine (5-Aza-dC), which is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells and restore the expression levels of their target genes in Jurkat cells. This result suggests that the overexpression of DNMTs could contribute to the development of T-cell leukemias by inducing hypermethylation of the target genes. Together, our results show that aberrant hypermethylation is an important molecular mechanism in the progression of T-cell leukemias, and thus could prove useful as a prognostic and/or diagnostic marker. Moreover, 5-Aza-dC might be a promising candidate for the treatment of T-cell leukemia.

PRSS8 methylation and its significance in esophageal squamous cell carcinoma.

Esophageal cancer is one of the most common cancers worldwide, and the incidence and mortality is increasing rapidly in recent years in China, but the underlying mechanisms are largely unclear. Herein we found that the expression of PRSS8, a serine protease prostasin, is significantly decreased in esophageal squamous cell carcinomas (ESCC) at mRNA and protein levels. The reduction of PRSS8 was well correlated with poor differentiation and shorter survival time. Interestingly, ESCC stromal expression of PRSS8 was significantly correlated with stromal lymphocyte infiltration and cancer progression. Methylation specific PCR showed that PRSS8 was hypermethylated in ESCC tissues and ESCC cell lines, which was linked to the downregulation of PRSS8 expression and decreased activities of PRSS8 promoter. De-methylation agent decitabine was able to restore PRSS8 expression, leading to the inhibition of cancer cell proliferation, motility, migration and cell cycle arrest. However, the restored PRSS8 and its tumor inhibition could be reversed by small interfering RNA targeting PRSS8. Mechanistic study showed that tumor inhibition of PRSS8 may be associated with proliferation- and epithelial mesenchymal transition - related proteins in ESCC cells. In conclusion, our finding showed that PRSS8 methylation and its stromal expression had important clinical significance in ESCC.

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

ABSTRACTAberrant DNA methylation is a critical feature of cancer. Epigenetic therapy seeks to reverse these changes to restore normal gene expression. DNA demethylating agents, including 5-aza-2′-deoxycytidine (DAC), are currently used to treat certain leukemias, and can sensitize solid tumors to chemotherapy and immunotherapy. However, it has been difficult to pin the clinical efficacy of these agents to specific demethylation events, and the factors that contribute to the durability of response remain largely unknown. Here we examined the genome-wide kinetics of DAC-induced DNA demethylation and subsequent remethylation after drug withdrawal in breast cancer cells. We find that CpGs differ in both their susceptibility to demethylation and propensity for remethylation after drug removal. DAC-induced demethylation was most apparent at CpGs with higher initial methylation levels and further from CpG islands. Once demethylated, such sites exhibited varied remethylation potentials. The most rapidly remethylating CpGs regained >75% of their starting methylation within a month of drug withdrawal. These sites had higher pretreatment methylation levels, were enriched in gene bodies, marked by H3K36me3, and tended to be methylated in normal breast cells. In contrast, a more resistant class of CpG sites failed to regain even 20% of their initial methylation after 3 months. These sites had lower pretreatment methylation levels, were within or near CpG islands, marked by H3K79me2 or H3K4me2/3, and were overrepresented in sites that become aberrantly hypermethylated in breast cancers. Thus, whereas DAC-induced demethylation affects both endogenous and aberrantly methylated sites, tumor-specific hypermethylation is more slowly regained, even as normal methylation promptly recovers. Taken together, these data suggest that the durability of DAC response is linked to its selective ability to stably reset at least a portion of the cancer methylome.

Aberrant DNA Methylation Important Factor in Intervertebral Disc Pathophysiology

Introduction Intervertebral disc (IVD) degeneration is one of the most important aging health problems and a leading cause of disability. However, little is known about pathophysiology of this condition. This study investigates the role of epigenetic factor in intervertebral disc gene regulation. Material and Methods IVD tissues were obtained from donors undergoing anterior lumbar discectomy procedures. The collected tissues were minced and immediately used for cell isolation by sequential enzyme digestion. The demethylation agent 5-azacytidine (5-AzaC) was used to inhibit DNA methylation in IVD cells. The cells were then analyzed for change in DNA methylation using Methyl-Profiler PCR array. We used Methyl-Profiler PCR array containing developmental and cell cycle regulatory genes. The Methyl-Profiler PCR arrays were performed as per manufacturer's instructions. Briefly, the cells were treated with and without 5-azaC for 4 days, and genomic DNA were isolated. Then the equal amounts of genomic DNA were overnight digested with methyl-specific restriction digestion enzyme, nonspecific restriction enzyme, a combination of methyl specific and non-specific, and a control mock digestion. Following digestion, real time PCR was performed using biorad-MyiQ cycler. The data was analyzed according to manufacturer's instructions. Results Methyl-Profiler PCR array showed novel genes hypo-methylated following 5-azacytidine treatment. We found that around 6 genes' promoters differentially methylated after experimental demethylation in IVD cells. The genes differentially methylated are CCND2, CDH1, HIC1, MGMT, PRDM2 and TP73. Real time PCR showed these genes were differentially regulated relative to control. Conclusion There are reports that these differentially methylated genes have a role in various age related degenerative diseases. We speculate that these genes may also play an important role in intervertebral disc degeneration. It would be interesting to investigate the expression of these genes and their methylation pattern in aging human degenerative IVD. In summary, our results suggest that demethylation of IVD cells may be one of the factors involved in IVD degeneration. Further studies are required to confirm our findings and to better understand the effects of aberrant DNA methylation on IVD biology and disease pathophysiology.

Methylation and expression of Epstein–Barr virus latent membrane protein 1, 2A and 2B in EBV-associated gastric carcinomas and cell lines

BackgroundPromoter CpG methylation of Epstein–Barr virus (EBV) genome plays an essential role in maintaining viral latency. Latent membrane protein (LMP) 1, 2A and 2B of EBV exert multiple oncogenic properties by activating multiple signal pathways and modulating the expression of various oncogenes. AimsTo study the methylation and expression of LMP1, 2A and LMP2B in EBV-positive cell lines and EBV-associated tumors. MethodsThe methylation profiles of LMP1p, LMP2Ap and LMP2Bp were evaluated by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP), as well as their expression by quantitative real-time (qRT)-PCR in 41 EBV-associated carcinomas (EBVaGCs) and 5 EBV-positive cell lines. ResultsAll LMP promoters were methylated at different degrees in EBV-positive cell lines and hypermethylated in EBV-associated gastric carcinomas, while unmethylated LMP2Ap alleles were detected in B95-8 cell line. Following 5-aza-2′-deoxycytidine (5-aza) treatment, the LMP1 expression was restored along with concomitant promoter demethylation; changes of LMP2A and LMP2B expression were different in different cells. ConclusionMethylation of LMP1, 2A and 2B promoters mediates the silencing of LMP1, 2A and 2B in EBV-associated carcinomas and cell lines in varying degrees, and could be reactivated by demethylation agent and thus may contribute to the therapy of EBVaGCs.

Immuno-regulated common markers but different network signatures in two associated cancers: evidences from epigenetic treatment.

Networks are now widely accepted inference tools in translational oncology. Besides providing agnostic model frameworks for complex data-driven clinical problems of diagnostic, therapeutic and prognostic impacts, networks mainly support insights, testable hypotheses and decision processes on the basis of their topological configurations and connectivity patterns. The purpose of this study is to emphasize the role of both gene and network signatures in two specific cancers. Retinoblastoma (RB) and osteosarcoma are associated to some extent. It is known that patients who carry germline mutations in the RB1 gene, and who survive RB, are typically at an increased risk of early-onset second cancers, including osteosarcomas. Gene signatures are widely used, but also criticized for their partial lack of reproducibility. Network signatures include gene association dynamics by identifying modules or communities in which subsets of genes functionally belong. Two cancer cell lines (one per cancer type) were subjected to a similar epigenetic treatment regimen, using a demethylation agent (DAC, and including similar dose and time course administration). A minimal set of shared differentially expressed (DEG) genes was identified in cancer-specific cell lines from microarray analyses. However, the identified immune signatures were observed to translate into much diversified network signatures. Our evidence is relevant to therapeutic developments, indicating that preference should be assigned to the assessment of bio-entities in a connected environment rather than considering single entities alone.

Cigarette Smoke-Induced Hypermethylation of the GCLC Gene Is Associated With COPD

Cigarette smoking is a major environmental contributor to COPD, but understanding its epigenetic regulation of oxidative genes involved in the pathogenesis of COPD remains elusive. We analyzed DNA methylation on glutamate-cysteine ligase catalytic subunit (GCLC), glutathione S-transferase M1 (GSTM1), glutathione S-transferase P1 (GSTP1), and superoxide dismutase 3 (SOD3) promoters in clinical samples from patients with COPD (current-smoker [CS-COPD]; ex-smoker [ES-COPD]) and subjects with normal pulmonary function (current-smoker [CS-NS]; ex-smoker [ES-NS]; never-smoker [NC]). Expression of GCLC messenger RNA (mRNA) and glutathione (GSH) synthesis in these clinical samples and human bronchial epithelial (BEAS-2B) cells stimulated by cigarette-smoke extract (CSE) was evaluated. GCLC mRNA and protein levels were measured to determine effects of demethylation and deacetylation agents on CSE-treated BEAS-2B cells. The DNA methylation level of the GCLC promoter was significantly increased in CS-COPD, CS-NS, and ES-COPD groups compared with ES-NS and NC groups. However, there were no significant differences in DNA methylation values of GSTM1, GSTP1, and SOD3 promoters among these groups. Expression of GCLC mRNA was downregulated in the lungs, and GSH levels decreased in plasma as a consequence of hypermethylation of the GCLC promoter. Similarly, CSE-treated BEAS-2B cells had hypermethylation of the GCLC gene, mRNA downregulation, and a decreased intracellular GSH level. GCLC expression in CSE-treated BEAS-2B cells was restored by the methylation inhibitor, 5-aza-2'-deoxycytidine, but not by the deacetylation agent, trichostatin A. Cigarette smoke-induced hypermethylation of the GCLC promoter is related to the initiation and progression of COPD. Our finding may provide a new strategy for COPD intervention by developing demethylation agents targeting GCLC hypermethylation.

Valproic acid and 5-azacytidine promote an increase of stemness phenotype in human osteosarcomas

Osteosarcoma (OS) is the most common pediatric tumour of bone in the world. It leads to local invasion and early metastasis to lung. The prognosis is poor with the 5-year survival rate of 65% for patients with primary tumour and 20% for patients with metastasis. OS contain a small cell population with stem cell like properties, referred to as cancer stem cells characterized by CD133 expression [1,2]. Recent studies have implicated epigenetic alterations, including DNA methylation and histone modifications, as prominent factors that contribute to the phenotype of cancer progenitor or stem cells. Here, we examined the epigenetic effects of the HDAC inhibitor valproic acid (VPA) and demethylating agent, 5’azacytidine (DAC), on the stemness phenotype in osteosarcoma cell lines. Saos-2 and MG63 cell lines were treated with 0,5 mM VPA and 3µM DAC for 48 hours alone, and in combination. CD133 expression as well as stemness markers expression including OCT4, Sox2 and Nanog was analyzed by flow cytometry and real-time PCR. Vimentin and osteocalcin levels were also tested. Sarcospheres formation rate was assessed as spheres number/seed single cells number. Specific histone modifications including H3-trymethyl-k9, H3-acetyl-k9, H3-trymethil-k27 and global methylation were analysed by flow cytometry and immunofluorescence. Moreover, soft agar and invasion assays were performed. Our findings indicated that DAC or VPA and their combination induced an increase of stemness characteristics of OS cells in terms of high expression of CD133, OCT4, Sox2 and Nanog and high sarcospheres-forming efficiency. Interestingly, combined treatment with DAC and VPA induced an increase of CD133 expression in a synergistic manner in all cell lines. Vimentin resulted up-regulated after treatment, whereas, the level of osteocalcin remained similar before and after treatment. Furthermore, soft agar assay revealed major colony-forming efficiency in treated cells compared to untreated cells, whereas invasion potential decreased after drug treatment. Interestingly, histone modification analyses correlated with those typical of embryonic stemness with an increase of H3-acetyl-k9 and decrease of H3-trymethyl-k9, and H3-trymethil-k27 after drug treatment as well as global methylation decreased in treated cells. In conclusion, DAC and VPA induced an increase of stemness associated to an decrease of global methylation, increase of acetylation and decrease of H3-trymethyl-k9, and H3-trymethil-k27. This study was supported by MIUR-EU funds (PON 01_02834 and PRIN 2011/13)

Expression of Cancer Testis Antigens in Colorectal Cancer: New Prognostic and Therapeutic Implications.

Background. While cancer/testis antigens (CTAs) are restricted in postnatal tissues to testes and germ line-derived cells, their role in cancer development and the clinical significance of their expression still remain to be better defined. Objective. The aim of this study was to investigate the level of CTA expression in colon samples from patients with colorectal cancer (CRC) in relation to patient clinical status. Methods. Forty-five patients with newly diagnosed colorectal cancer were included in the study. We selected a panel of 18 CTAs that were previously detected in CRC as well as some new gene candidates, and their expression was detected at the mRNA level by employing RQ-PCR. Additionally, we evaluated CTA expression in three colon cancer cell lines (CL-188, HTB-39, and HTB-37) after exposure to the DNA methylation-modifying drug 5-azacytidine. Results. We report that 6 out of 18 (33%) CTAs tested (MAGEA3, OIP5, TTK, PLU1, DKKL1, and FBXO39) were significantly (p < 0.05) overexpressed in tumor tissue compared with healthy colon samples isolated from the same patients. Conclusions. Moreover, we found that MAGEA3, PLU-1, and DKKL expression positively correlated with disease progression, evaluated according to the Dukes staging system. Finally, 5-azacytidine exposure significantly upregulated expression of CTAs on CRC cells, which indicates that this demethylation agent could be employed therapeutically to enhance the immune response against tumor cells.

Epigenetic DNA Methylation Linked to Social Dominance.

Social status hierarchies are ubiquitous in vertebrate social systems, including humans. It is well known that social rank can influence quality of life dramatically among members of social groups. For example, high-ranking individuals have greater access to resources, including food and mating prerogatives that, in turn, have a positive impact on their reproductive success and health. In contrast low ranking individuals typically have limited reproductive success and may experience lasting social and physiological costs. Ultimately, social rank and behavior are regulated by changes in gene expression. However, little is known about mechanisms that transduce social cues into transcriptional changes. Since social behavior is a dynamic process, we hypothesized that a molecular mechanism such as DNA methylation might play a role these changes. To test this hypothesis, we used an African cichlid fish, Astatotilapia burtoni, in which social rank dictates reproductive access. We show that manipulating global DNA methylation state strongly biases the outcomes of social encounters. Injecting DNA methylating and de-methylating agents in low status animals competing for status, we found that animals with chemically increased methylation states were statistically highly likely to ascend in rank. In contrast, those with inhibited methylation processes and thus lower methylation levels were statistically highly unlikely to ascend in rank. This suggests that among its many roles, DNA methylation may be linked to social status and more generally to social behavior.

Quantification for total demethylation potential of environmental samples utilizing the EGFP reporter gene

The demethylation potential of pollutants is arguably an innate component of their toxicity in environmental samples. A method was developed for determining the total demethylation potential of food samples (TDQ). The demethylation epigenetic toxicity was determined using the Hep G2 cell line transfected with pEGFP-C3 plasmids containing a methylated promoter of the EGFP reporter gene. The total demethylation potential of the sample extracts (the 5-AZA-CdR demethylation toxic equivalency) can be quantified within one week by using a standard curve of the 5-AZA-CdR demethylation agent. To explore the applicability of TDQ for environmental samples, 17 groundwater samples were collected from heavy polluted Kuihe river and the total demethylation potentials of the sample extracts were measured successfully. Meaningful demethylation toxic equivalencies ranging from 0.00050 to 0.01747μM were found in all groundwater sample extracts. Among 19 kinds of inorganic substance, As and Cd played important roles for individual contribution to the total demethylation epigenetic toxicity. The TDQ assay is reliable and fast for quantifying the DNA demethylation potential of environmental sample extracts, which may improve epigenetic toxicity evaluations for human risk assessment, and the consistent consuming of groundwater alongside the Kuihe river pose unexpected epigenetic health risk to the local residents.

DNA Methylation in the Exon 1 Region and Complex Regulation of Twist1 Expression in Gastric Cancer Cells.

Twist1 overexpression is frequently observed in various cancers including gastric cancer (GC). Although DNA methylation of the Twist1 gene has been reported in cancer cells, the mechanisms underlying transcriptional activation remain uncertain. In this study, we first examined epigenetic alterations of the Twist1 using Twist1 transcription-positive and -negative cell lines that are derived from our established diffuse-type GC mouse model. Treatment with a DNA demethylation agent 5-aza-dC re-activated Twist1 expression in Twist1 expression-negative GC cells. According to methylation-specific PCR and bisulfite sequencing analysis, methylation at the CpG-rich region within Twist1 coding exon 1, rather than its promoter region, was tightly linked to transcriptional silencing of the Twist1 expression in mouse GC cells. Chromatin immunoprecipitation assays revealed that active histone mark H3K4me3 was enriched in Twist1 expression-positive cells, and inactive histone mark H3K9me3 was enriched in Twist1 expression-negative cells. The expression levels of Suv39h1 and Suv39h2, histone methyltransferases for H3K9me3, were inversely correlated with Twist1 expression, and knockdown of Suv39h1 or Suv39h2 induced Twist1 expression. Moreover, Sp1 transcription factor bound to the exon 1 CpG-rich region in Twist1 expression-positive cell lines, and Twist1 expression was diminished by mithramycin, which that interferes with Sp1 binding to CpG-rich regulatory sequences. Our studies suggested that the Twist1 transcription in GC cells might be regulated through potential cooperation of DNA methylation, histone modification in complex with Sp1 binding to CpG-rich regions within the exon 1 region.

WITHDRAWN: Ski is silenced by methylation and acts as tumor suppressor in non-small cell lung cancer

// Mian Xie 1 , Xiaojun Wu 2 , Chaosheng He 3 , Jiexia Zhang 1 , Jinjun Zhang 4 1 China State Key Laboratory of Respiratory Disease and Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 2 State Key Laboratory of Oncology in Southern China, Department of Colorectal Surgery, Sun Yat-Sen University Cancer Center, Guangzhou, China 3 Department of Internal Medicine, Guangdong General Hospital, Guangzhou, China 4 Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China Correspondence to: Mian Xie, e-mail: mianxie@gird.cn Keywords: lung cancer, Ski, DNA methylation, prognosis Received: July 20, 2015&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: November 22, 2015&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: December 12, 2015 ABSTRACT Epigenetic silencing of tumor suppressors contributes to the development and progression of lung cancer. We recently found that Ski was hypermethylated in lung cancer. This study aimed to clarify its epigenetic alteration, molecular mechanisms and clinical significance in lung cancer. Ski methylation was evaluated by methylation-specific PCR (MS-PCR) and bisulfite sequencing. mRNA level of Ski was measured by RT-PCR and compared with the methylation status. Ski methylation correlated with decreased mRNA expression in human lung cancer cell lines. Ski hypermethylation was detected in 56.0% of primary lung tumors and associated with poor differentiation and late tumor stage. Demethylation agent 5-aza-2&prime;-deoxycytidine (5-aza-2&prime;dC) restored Ski expression. Re-expression of Ski in lung cancer cells inhibited cell proliferation, clonogenicity, migration, invasion and tumor formation. Ski decreased transcriptional activities of Smads and TAZ. Multivariate analysis showed that patients with Ski positive expression had a better overall survival in resected non-small cell lung cancer (NSCLC) patients. Our results revealed that Ski acts as a tumor suppressor inactivated by DNA methylation and is an independent prognostic factor of lung cancer.

Landscape of DNA Methylation and Genetic Profiles in 291 Patients with Myelodysplastic Syndromes

DNA hypermethylation has long been implicated in the pathogenesis of myelodysplastic syndromes (MDS) and also highlighted by the frequent efficacy of demethylating agents to this disease. Meanwhile, recent genetic studies in MDS have revealed high frequency of somatic mutations involving epigenetic regulators, suggesting a causative link between gene mutations and epigenetic alterations in MDS. The accumulation of genetic and epigenetic alterations promotes tumorigenesis, hypomethylating agents such as Azacitidine exert their therapeutic effect through inhibition of DNA methylation. However, the relationship between patterns of epigenetic phenotypes and mutations, as well as their impact on therapy, has not been clarified.To address this issue, we performed genome-wide DNA methylation profiling (Infinium 450K) in combination with targeted-deep sequencing of 104 genes for somatic mutations in 291 patients with MDS. Beta-mixture quantile normalization was performed for correcting probe design bias in Illumina Infinium 450k DNA methylation data. Of the >480,000 probes on the methylation chip, we selected probes using the following steps: (i) probes annotated with "Promotor_Associated" or "Promoter_Associated_Cell_type_specific; (ii) probes designed in "Island", "N_Shore" or "S_Shore"; (iii) removing probes designed on the X and Y chormosomes; (iv) removing probes with >10% of missing value. Consensus clustering was performed utilizing the hierarchical clustering based on Ward and Pearson correlation algorithms with 1000 iterations on the top 0.5% (2,000) of probes showing high variation by median absolute deviation across the dataset using Bioconductor package Consensus cluster plus. The number of cluster was determined by relative change in area under cumulative distribution function curve by consensus clustering.Unsupervised clustering analysis of DNA methylation revealed 3 subtypes of MDS, M1-M3, showing discrete methylation profiles with characteristic gene mutations and cytogenetics. The M1 subtype (n=121) showed a high frequency of SF3B1 mutations, exhibiting the best clinical outcome, whereas the M2 subtype (n=106), characterized by frequent ASXL1, TP53 mutations and high-risk cytogenetics, showed the shortest overall survival with the hazard ratios of 3.4 (95% CI:1.9-6.0) and 2.2 (95% CI:1.2-4.0) compared to M1 and M3, respectively. Finally, the M3 subtype (n=64) was highly enriched (70% of cases) for biallelic alterations of TET2 and showed the highest level of CpG island methylation and showed an intermediate survival.In the current cohort, we had 47 patients who were treated with demethylating agents, including 11 responders and 36 non-responders. When DNA methylation status at diagnosis was evaluated in terms of response to demethylating agents, we identified 54 differentiated methylated genes showing >20% difference in mean methylation levels between responders and non-responders (q < 0.1). Twenty-five genes more methylated in responders were enriched in functional pathways such as chemokine receptor and genes with EGF-like domain, whereas 29 less methylated gene in responders were in the gene set related to regulation of cell proliferation. Genetic alterations were also assessed how they affected treatment responses. In responders, TET2 mutated patients tended to more frequently respond (45% vs 34%), whereas patients with IDH1/2 and DNMT3A mutations were less frequently altered (0% vs 14%, 9% vs 14%) in responders, compared in non-responders.In conclusion, our combined genetic and methylation analysis unmasked previously unrecognized associations between gene mutations and DNA methylation, suggesting a causative link in between. We identified correlations between genetic/epigenetic profiles and the response to demethylating agents, which however, needs further investigation to clarify the mechanism of and predict response to demethylation agents in MDS. DisclosuresAlpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kiyoi:Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.k.: Research Funding; Pfizer Inc.: Research Funding; Takeda Pharmaceutical Co.,Ltd.: Research Funding; MSD K.K.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Alexion Pharmaceuticals.: Research Funding; Teijin Ltd.: Research Funding; Zenyaku Kogyo Company,Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Patents & Royalties, Research Funding; Nippon Shinyaku Co.,Ltd.: Research Funding; Japan Blood Products Organization.: Research Funding; Eisai Co.,Ltd.: Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Kyowa-Hakko Kirin Co.,Ltd.: Consultancy, Research Funding; Fujifilm Corporation.: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Bristol-Myers Squibb.: Research Funding; Chugai Pharmaceutical Co.,LTD.: Research Funding; Mochida Pharmaceutical Co.,Ltd.: Research Funding. Kobayashi:Gilead Sciences: Research Funding. Naoe:Toyama Chemical CO., LTD.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Celgene K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Miyazaki:Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Kyowa-Kirin: Honoraria, Research Funding.

PU.1-Induced IRF4 Down-Regulation and Subsequent IRF7 up-Regulation in Myeloma Cells

PU.1 is an Ets family transcription factor, which is necessary for differentiation of both myeloid and lymphoid lineages. It was previously reported that conditional knockout of the upstream regulatory element (URE) located in 14 kb 5' of the PU.1 gene resulted in down-regulation of PU.1 expression in granulocytes and B lymphocytes by 80% compared to that of wild type and induced acute myeloid leukemia and CLL-like diseases in mice. Since the URE contains a suppressor region for PU.1 expression in T cells, such mice express PU.1 in T cells and develop T cell lymphoma. Thus, the failure of proper expression of PU.1 in certain differentiation stages in certain cell lineages appears to result in hematological malignancies. We previously reported that PU.1 is down-regulated in various myeloma cell lines. In addition, PU.1 is expressed in normal plasma cells and PU.1 is down-regulated in myeloma cells of certain myeloma patients, who appear to have poor prognosis. In those myeloma cell lines, the promoter and URE of the PU.1 gene are highly methylated. A demethylation agent, 5-aza-2'-deoxycytidine, induced PU.1 up-regulation, growth arrest, and apoptosis in myeloma cell lines, KMS12PE and KHM11. In addition, conditionally expressed PU.1 induced cell growth arrest and apoptosis in PU.1-low-negative myeloma cell lines, U266 and KMS12PE, suggesting that PU.1 is a tumor suppressor for myeloma cells. To elucidate the mechanisms of the cell growth arrest and apoptosis in myeloma cells induced by PU.1, we performed DNA microarray analysis to compare gene expression levels before and after PU.1 expression. Among cell-cycle related genes, p21WAF1/CIP1 was found up-regulated in U266 cells, while among apoptosis related genes, TRAIL was highly up-regulated in both U266 and KMS12PE cell lines. With further investigation, we concluded that PU.1 directly transactivated the TRAIL gene in myeloma cells, leading to apoptosis.Based on the DNA microarray data generated, we found that IRF4 is downregulated in U266 myeloma cells after PU.1 induction. It has been reported that knockdown of IRF4 induces apoptosis in myeloma cell lines. Therefore, we examined whether IRF4 was down-regulated in three myeloma cell lines, U266, KMS12PE, and KHM11 following PU.1 induction. Conditional expression of PU.1 by tet-off system induced IRF4 down-regulation in U266and KMS12PE cells. With lentiviral transduction method, ectopic expression of PU.1 also induced IRF4 down-regulation, cell-cycle arrest, and apoptosis in KHM11 cells. To investigate the role of IRF4 in PU.1-expressing U266 cells, we stably expressed IRF4, partially rescuing U266 cells from apoptosis. IRF4 is known to directly bind to the IRF7 promoter and down-regulate IRF7 expression in activated B cell-like (ABC) subtype of diffuse large B-cell lymphoma cells. Therefore, we examined whether IRF4 bound to the IRF7 promoter in KMS12PEand U266cells using chromatin immunoprecipitation assays. We found that IRF4 directly bound to the IRF7 promoter in both myeloma cell lines. When we overexpressed PU.1, IRF4 levels were decreased and the IRF4 binding to the IRF7 promoter was significantly reduced in those cell lines. Moreover, knockdown of IRF7 significantly rescued PU.1-expressing U266cells from apoptosis. These data strongly suggest that PU.1-induced apoptosis is associated with IRF4 down-regulation and subsequent IRF7 up-regulation in myeloma cells. Since IRF4 is essential transcription factor for myeloma cell survival, up-regulation of PU.1 by demethylation agents, including 5-aza-2'-deoxycytidine may serve as a promising therapeutic modality of multiple myeloma by inducing down-regulation of IRF4. DisclosuresNo relevant conflicts of interest to declare.

TET2 Downregulation Leads to AML Cells Sensitive to Decitabine

Background: Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and have very lethal rate. Chemotherapy is the main method to treat AML, but the complete remission rate is still not very optimal. With the development of genetic and molecular biology technologies, more and more molecular biomarkers are found, some of them are useful for us to evaluate the prognosis and can help us to tailor the treatment plan for different patients. TET2, a member of the ten-eleven-translocation(TET) family genes which can modify DNA by catalyzing the conversion of 5mehtyl-cytosine(5-mC) to 5-hydroxymethyl-cytosine(5-hmC), is often inactivated through loss-of-function mutation and deletion in myeloid malignancies. Recent clinical research reported that the lower the expression of TET2 in MDS and AML patients, the better the response to decitabine (DAC, a demethylation agent) will be. However, the mechanism of the phenomenon is still unknown. Our investigation is trying to uncover the mechanism how TET2 protein levels are negatively related with AML sensitivity to decitabine.Methods: We detected TET2 mRNA expression level in acute leukemia cell lines, bone marrow AML specimens and peripheral blood mononuclear cells from healthy donors by semiquantitative real time polymerase chain reaction (qRT-PCR). Western blot is also applied to detect TET2 protein expression. In order to access TET2 methylation status, we used the methylation-specific PCR. And we also checked the mutant status of TET2 in U937 and KG-1 cell line. CCK8 and flow cytometry are used to detect cell proliferation rate, cell apoptosis, and cell cycle profile. Also, we developed TET2 knock-down and overexpression lentivirus to transfect AML cell lines to explore the mechanism why TET2 expression level is related to the response of DAC. Last, gene array is used to compare gene expression level changes between TET2 knock-down cell lines (or TET2 overexpression cell lines) and the control cell lines.Results: The AML cell lines (KG-1, U937, Kasumi, HL-60, THP-1) and AML patients specimens express lower TET2 than that of PBMC from the healthy donor (P<0.05). Among AML cell lines, U937 barely expresses TET2, while KG-1 expresses TET2 relatively higher than other AML cell lines. The methylation-specific PCR showed that TET2 in U937 was partially methylated while KG-1 was not. After using decitabine to treat U937 cell line, the TET2 methylation status was attenuated. And all the exons of TET2 were not detected any mutation in KG-1 AND U937. Then, we used CCK8 to compare the response difference to DAC between U937 and KG-1 and found that U937 is much more sensitive to DAC rather than KG-1 (P<0.05). Next, we constructed a TET2 shRNA to transfect KG-1, both qRT-PCR and WB were used to verification the knock-down efficiency. Again, CCK8 told us that KG-1 TET2 knock-down cells was more sensitive to DAC than KG-1 NC cells. Flow cytometry identified that cell cycle profile were altered between KG-1 TET2 knock-down cells and KG-1 NC cells. Gene array (KG-1 TET2 KD and KG-1 NC) showed that the expression levels of cell cycle related genes (e.g. CCNB2,RBL1), DNA replication related genes (e.g. PRIM1, RCF3, FEN1) and many other function genes were changed between the knock-down and control cell line.Conclusion: Our study showed that the sensitivity to decitabine of AML cell lines is related to TET2 expression level, knock-down TET2 in KG-1 can increase its vulnerability to decitbine. And the mechanism may be related to the changing expression levels of the genes which regulating cell cycles and DNA replication. DisclosuresNo relevant conflicts of interest to declare.

Pharmacological DNA demethylation: Implications for cancer immunotherapy

ABSTRACTRecent studies have demonstrated that DNA demethylation agents can mimic a viral infection by induction of dsRNAs. This viral mimicry leads to an antiviral response mediated by the cytosolic pattern recognition receptor MDA5, followed by MAVS (IPS1) activation, IRF7 nuclear translocation and upregulation of type III Interferon and interferon-stimulated genes.