DNA Hypermethylation Of CpG Islands Research Articles

Cancer-associated DNA hypermethylation of Polycomb targets requires DNMT3A dual recognition of histone H2AK119 ubiquitination and the nucleosome acidic patch.

During tumor development, promoter CpG islands that are normally silenced by Polycomb repressive complexes (PRCs) become DNA-hypermethylated. The molecular mechanism by which de novo DNA methyltransferase(s) [DNMT(s)] catalyze CpG methylation at PRC-regulated regions remains unclear. Here, we report a cryo-electron microscopy structure of the DNMT3A long isoform (DNMT3A1) amino-terminal region in complex with a nucleosome carrying PRC1-mediated histone H2A lysine-119 monoubiquitination (H2AK119Ub). We identify regions within the DNMT3A1 amino terminus that bind H2AK119Ub and the nucleosome acidic patch. This bidentate interaction is required for effective DNMT3A1 engagement with H2AK119Ub-modified chromatin in cells. Further, aberrant redistribution of DNMT3A1 to Polycomb target genes recapitulates the cancer-associated DNA hypermethylation signature and inhibits their transcriptional activation during cell differentiation. This effect is rescued by disruption of the DNMT3A1-acidic patch interaction. Together, our analyses reveal a binding interface critical for mediating promoter CpG island DNA hypermethylation, a major molecular hallmark of cancer.

Deficiency of proline/serine-rich coiled-coil protein 1 (PSRC1) accelerates trimethylamine N-oxide-induced atherosclerosis in ApoE−/− mice

AimsThe main therapeutic strategies for coronary artery disease (CAD) are mainly based on the correction of abnormal cholesterol levels; however, residual risks remain. The newly proven gut microbial metabolite trimethylamine N-oxide (TMAO) linked with CAD has broadened our horizons. In this study, we determined the role of proline/serine-rich coiled-coil protein 1 (PSRC1) in TMAO-driven atherosclerosis. Methods and resultsWe first analyzed the levels of TMAO and PSRC1 in patients with or without atherosclerosis with a target LDL-C < 1.8 mmol/L. Plasma TMAO levels were increased and negatively associated with decreased PSRC1 in peripheral blood mononuclear cells. Animals and in vitro studies showed that TMAO inhibited macrophage PSRC1 expression due to DNA hypermethylation of CpG islands. ApoE−/− mice fed a choline-supplemented diet exhibited reduced PSRC1 expression accompanied by increased atherosclerotic lesions and plasma TMAO levels. We further deleted PSRC1 in apoE−/− mice and PSRC1 deficiency significantly accelerated choline-induced atherogenesis, characterized by increased macrophage infiltration, foam cell formation and M1 macrophage polarization. Mechanistically, we overexpressed and knocked out PSRC1 in cultured macrophages to explore the mechanisms underlying TMAO-induced cholesterol accumulation and inflammation. PSRC1 deletion impaired reverse cholesterol transport and enhanced cholesterol uptake and inflammation, while PSRC1 overexpression rescued the proatherogenic phenotype observed in TMAO-stimulated macrophages, which was partially attributed to sulfotransferase 2B1b (SULT2B1b) inhibition. ConclusionsHerein, clinical data provide evidence that TMAO may participate in the development of CAD beyond well-controlled LDL-C levels. Our work also suggests that PSRC1 is a negative regulator mediating the unfavorable effects of TMAO-containing diets. Therefore, PSRC1 overexpression and reduced choline consumption may further alleviate atherosclerosis.

Genome-Scale Methylation Analysis of Circulating Cell-Free DNA in Gastric Cancer Patients.

Aberrant DNA hypermethylation of CpG islands (CGIs) occurs frequently and is genome-wide in human gastric cancer (GC). A DNA methylation approach in plasma cell-free DNA (cfDNA) is attractive for the noninvasive detection of GC. Here, we performed genome-scale cfDNA methylation analysis in patients with GC. We used MCTA-Seq, a genome-scale DNA methylation analysis method, on the plasma samples of patients with GC (n = 89) and control participants (n = 82), as well as 28 pairs of GC and adjacent noncancerous tissues. The capacity of the method for detecting GC and discriminating GC from colorectal cancer (CRC) and hepatocellular carcinoma (HCC) was assessed. We identified 153 cfDNA methylation biomarkers, including DOCK10, CABIN1, and KCNQ5, for detecting GC in blood. A panel of these biomarkers gave a sensitivity of 44%, 59%, 78%, and 100% for stage I, II, III, and IV tumors, respectively, at a specificity of 92%. CpG island methylation phenotype (CIMP) tumors and NON-CIMP tumors could be distinguished and detected effectively. We also identified several hundreds of cfDNA biomarkers differentially methylated between GC, CRC, and HCC, and showed that MCTA-Seq can discriminate early-stage GC, CRC, and HCC in blood by using a high specificity (approximately 100%) algorithm. Our comprehensive analyses provided valuable data on cfDNA methylation biomarkers of GC and showed the promise of cfDNA methylation for the blood-based noninvasive detection of GC.

GSTP1 positive prostatic adenocarcinomas are more common in Black than White men in the United States.

GSTP1 is a member of the Glutathione-S-transferase (GST) family silenced by CpG island DNA hypermethylation in 90-95% of prostate cancers. However, prostate cancers expressing GSTP1 have not been well characterized. We used immunohistochemistry against GSTP1 to examine 1673 primary prostatic adenocarcinomas on tissue microarrays (TMAs) with redundant sampling from the index tumor from prostatectomies. GSTP1 protein was positive in at least one TMA core in 7.7% of cases and in all TMA cores in 4.4% of cases. The percentage of adenocarcinomas from Black patients who had any GSTP1 positive TMA cores was 14.9%, which was 2.5 times higher than the percentage from White patients (5.9%; P < 0.001). Further, the percentages of tumors from Black patients who had all TMA spots positive for GSTP1 (9.5%) was 3-fold higher than the percentage from White patients (3.2%; P<0.001). In terms of association with other molecular alterations, GSTP1 positivity was enriched in ERG positive cancers among Black men. By in situ hybridization, GSTP1 mRNA expression was concordant with protein staining, supporting the lack of silencing of at least some GSTP1 alleles in GSTP1-positive tumor cells. This is the first report revealing that GSTP1-positive prostate cancers are substantially over-represented among prostate cancers from Black compared to White men. This observation should prompt additional studies to determine whether GSTP1 positive cases represent a distinct molecular subtype of prostate cancer and whether GSTP1 expression could provide a biological underpinning for the observed disparate outcomes for Black men.

Bacterial-Driven Inflammation and Mutant BRAF Expression Combine to Promote Murine Colon Tumorigenesis That Is Sensitive to Immune Checkpoint Therapy.

Colorectal cancer is multifaceted, with subtypes defined by genetic, histologic, and immunologic features that are potentially influenced by inflammation, mutagens, and/or microbiota. Colorectal cancers with activating mutations in BRAF are associated with distinct clinical characteristics, although the pathogenesis is not well understood. The Wnt-driven multiple intestinal neoplasia (MinApcΔ716/+) enterotoxigenic Bacteroides fragilis (ETBF) murine model is characterized by IL17-dependent, distal colon adenomas. Herein, we report that the addition of the BRAF V600E mutation to this model results in the emergence of a distinct locus of midcolon tumors. In ETBF-colonized BRAF V600E Lgr5 CreMin (BLM) mice, tumors have similarities to human BRAF V600E tumors, including histology, CpG island DNA hypermethylation, and immune signatures. In comparison to Min ETBF tumors, BLM ETBF tumors are infiltrated by CD8+ T cells, express IFNγ signatures, and are sensitive to anti-PD-L1 treatment. These results provide direct evidence for critical roles of host genetic and microbiota interactions in colorectal cancer pathogenesis and sensitivity to immunotherapy. SIGNIFICANCE: Colorectal cancers with BRAF mutations have distinct characteristics. We present evidence of specific colorectal cancer gene-microbial interactions in which colonization with toxigenic bacteria drives tumorigenesis in BRAF V600E Lgr5 CreMin mice, wherein tumors phenocopy aspects of human BRAF-mutated tumors and have a distinct IFNγ-dominant immune microenvironment uniquely responsive to immune checkpoint blockade.This article is highlighted in the In This Issue feature, p. 1601.

Abstract 4702: DNA methylation enhances apoptosis resistance of renal cancer cells through the control of mitochondria related gene expression

Abstract Many gene alterations have been identified in the onset of renal carcinogenesis. However, molecular alterations might be added to renal cancer cells during the progression. In order to uncover additional molecular alterations, we established highly malignant cancer cells which possess identical background to original cancer cells, and analyzed them in the present study. Since our previous works demonstrated that interaction with cancer microenvironment aids cancer cells with malignant trait through “education” and “selection”, serial orthotopic transplantation was utilized. First, parental human clear cell renal cell carcinoma cells, OS-RC-2, were subjected to serial orthotopic transplantation. Then, novel derivatives were obtained from resulting primary renal tumors. These derivatives formed primary renal tumor faster than parental cells and exhibited increased lung metastasis, suggesting that these derivatives were defined as “highly malignant cancer cells”. Next, we tried to identify the underlying molecular mechanism(s) for renal cancer progression through the characterization of highly malignant cancer cells. Gene expressions in each cell were profiled using RNA-sequencing, and these analyses revealed that expression of several genes associated with mitochondrial functions were dramatically down-regulated in highly malignant cancer cells. Since mitochondrial electron transport chain triggers apoptotic cell death, decrease of these genes may contribute to suppression of apoptosis of highly malignant cancer cells. Cell culture and in vivo experiments demonstrated that mitochondrial dysfunction is responsible for increased survival of renal cancer cells and cancer metastasis. Finally, regulatory mechanism of downregulation of these mitochondria-related genes was analyzed. Bisulfite sequencing analysis revealed that the downregulation may occur by DNA hypermethylation of CpG islands in the promoter regions in renal cancer cells.Taken together, these findings suggested that renal cancer cells acquire additional anti-apoptotic ability through the DNA methylation during the interactions with renal microenvironment, which may in turn lead to the cancer progression and metastasis. We are currently investigating whether the effect of anti-cancer reagent is enhanced in the presence of DNA methylation inhibitors. Citation Format: Kosuke Miyakuni, Jun Nishida, Shogo Ehata, Kohei Miyazono. DNA methylation enhances apoptosis resistance of renal cancer cells through the control of mitochondria related gene expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4702.

Hypermethylation of Corticotropin Releasing Hormone Receptor-2 Gene in Ulcerative Colitis Associated Colorectal Cancer.

The difficulty of early diagnosis of colitis associated colorectal cancer (CACRC) due to colonic mucosal changes in long-standing ulcerative colitis (UC) patients is often experienced in daily clinical practice. Noninvasive objective monitoring for cancer development is advantageous for optimizing treatment strategies in UC patients. We aimed to examine the epigenetic alterations occurring in CACRC, focusing on DNA hypermethylation of CpG islands. The level of DNA methylation in CpG cites was compared between CACRC and the counterpart non-tumorous mucosa using Infinium HumanMethylation 450K BeadChip. Our subjects included 3 males and 3 females (median age, 49.5 years). The 450K CpG site DNA methylation microarray revealed that the difference in β value (level of hypermethylation) was the highest for corcicotropin releasing hormone receptor 2 (CRHR2) between CACRC and counterpart non-tumorous mucosa. Detection of hypermethylation of CRHR2 may be promising for cancer screening in UC patients.

Detection of Colorectal Cancer in Circulating Cell-Free DNA by Methylated CpG Tandem Amplification and Sequencing.

Aberrant DNA hypermethylation of CpG islands occurs frequently throughout the genome in human colorectal cancer (CRC). A genome-wide DNA hypermethylation analysis technique using circulating cell-free DNA (cfDNA) is attractive for the noninvasive early detection of CRC and discrimination between CRC and other cancer types. We applied the methylated CpG tandem amplification and sequencing (MCTA-Seq) method, with a fully methylated molecules algorithm, to plasma samples from patients with CRC (n = 147) and controls (n = 136), as well as cancer and adjacent noncancerous tissue samples (n = 66). We also comparatively analyzed plasma samples from patients with hepatocellular carcinoma (HCC; n = 36). Dozens of DNA hypermethylation markers including known (e.g., SEPT9 and IKZF1) and novel (e.g., EMBP1, KCNQ5, CHST11, APBB1IP, and TJP2) genes were identified for effectively detecting CRC in cfDNA. A panel of 80 markers discriminated early-stage CRC patients and controls with a clinical sensitivity of 74% and clinical specificity of 90%. Patients with early-stage CRC and HCC could be discriminated at clinical sensitivities of approximately 70% by another panel of 128 markers. MCTA-Seq is a promising method for the noninvasive detection of CRC.

Abstract PL02-04: Genomic and epigenomic studies of cholangiocarcinoma in diverse populations

Abstract It is well-known that Asian prevalent cancers are often associated with a pathogen or carcinogen. For example, hepatocellular carcinoma is driven by hepatitis B and C viruses, gastric cancer by Helicobacter pylori, and nasopharyngeal cancer by Epstein-Barr virus. In this presentation, we focus on biliary tract cancer or cholangiocarcinoma (CCA) of different etiologies which are associated with chronic carcinogenic exposures leading to distinct genomic and epigenomic changes. CCA is a malignant tumor of bile duct epithelial cells with very poor prognosis which accounted for approximately 5% of all primary liver cancers diagnosed worldwide. It is broadly divided into intrahepatic, perihilar, and distal CCAs which may differ in their spectrum and frequency of genetic alterations, possibly reflecting differences in cell of origin and tumorigenic process. CCA displays distinct molecular mechanisms that require different therapeutic regimes for effective treatment representing an important unmet clinical need. Similarly, the genomic and epigenomic changes may serve as effective biomarkers for disease detection and monitoring. Risk factors for CCA are related to geography and etiology. In Western countries, primary sclerosing cholangitis (PSC) is the most common risk factor of CCA, and other risk factors include hepatolithiasis, choledochal cysts, cirrhosis, diabetes, obesity, alcohol consumption and smoking. On the other hand, in the northeast region of Thailand, Laos and Cambodia, infestation of liver fluke called Opisthorchis viverrini (OV) has been associated with the highest incidence of CCA, with approximately 20,000 cases annually. Once ingested, OV can inhabit the biliary tract within the human host for over 10 years, promoting carcinogenesis potentially via 1) mechanical damage to the biliary epithelia caused by the feeding activities of the parasites, 2) immunopathology because of infection-related inflammation, 3) the toxic effects of parasite excretory/secretory molecules and 4) the disruption of the bile homeostasis. Furthermore, we and others have recently identified a potentially new risk factor for CCA, especially in China, Taiwan, South Korea and Singapore. Through mutation signature analysis on somatic genomic data, we observed an association of CCA with the herbal carcinogen Aristolochic Acid (AA), which is a natural compound exclusively found in plants of the Aristolochia genus used in traditional remedies for numerous indications. With the goal of better understanding the underlying oncogenic mechanisms in CCA, we characterized and compared the genetic and epigenetic changes in OV and non-OV driven and AA-related CCAs from different populations. We found that in non-OV driven CCA, mutations in epigenetic modifier genes including IDH1/2 and BAP1 are the main drivers whereas OV-infected tumors are characterized by frequent p53 mutations. These mutational differences set the stage for more in-depth epigenomic characterization and mechanistic dissection of the tumorigenic process. Our recent data indicate that OV driven CCAs display predominantly CpG Island DNA hypermethylation whereas non-OV driven CCAs are predominantly CpG Shore DNA hypermethylated. OV and non-OV CCAs also differ in their enhancer landscape: OV CCAs exhibit widespread activation of inflammatory response genes, while genes involved in maintenance of cell junction are disrupted in non-OV CCAs. Several lines of evidence also support that chronic inflammation can lead to aberrant DNA methylation and de novo enhancer activity. Consequently, chronic inflammation associated with OV infection may play a key role in setting up divergent enhancer landscapes between OV-related and non-OV CCAs. Other than OV and non-OV driven CCAs, AA represents a profound public health risk in Asia due to its wide use in herbal remedies. We found that AA exposure causes unique mutational signature of A:T to T:A transversion, with an antisense strand bias. We recently detected AA signature in many CCA cases, especially from Asian countries. At present, we are conducting epigenomics studies on AA-related CCAs to understand how AA may impact the epigenomic landscape of CCA. Our multi-omics analysis reflects the contrasting etiologies of CCA, strongly positing that the same disease manifestation can be in fact driven by vastly different molecular circuitries and that each patient should be treated with appropriately targeted therapy. These findings will deepen our understanding of early transformation events in CCA, which may lead to the development of relevant biomarkers and treatment regimens for each subgroup of CCA. As such, our study illustrates the value of investigating specific cancers in the context of diverse populations and etiologies. Citation Format: Bin Tean Teh. Genomic and epigenomic studies of cholangiocarcinoma in diverse populations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr PL02-04.

DNA methyltransferase inhibition reduces inflammation-induced colon tumorigenesis

ABSTRACTChronic inflammation is strongly associated with an increased risk of developing colorectal cancer. DNA hypermethylation of CpG islands alters the expression of genes in cancer cells and plays an important role in carcinogenesis. Chronic inflammation is also associated with DNA methylation alterations and in a mouse model of inflammation-induced colon tumorigenesis, we previously demonstrated that inflammation-induced tumours have 203 unique regions with DNA hypermethylation compared to uninflamed epithelium. To determine if altering inflammation-induced DNA hypermethylation reduces tumorigenesis, we used the same mouse model and treated mice with the DNA methyltransferase (DNMT) inhibitor decitabine (DAC) throughout the tumorigenesis time frame. DAC treatment caused a significant reduction in colon tumorigenesis. The tumours that did form after DAC treatment had reduced inflammation-specific DNA hypermethylation and alteration of expression of associated candidate genes. When compared, inflammation-induced tumours from control (PBS-treated) mice were enriched for cell proliferation associated gene expression pathways whereas inflammation-induced tumours from DAC-treated mice were enriched for interferon gene signatures. To further understand the altered tumorigenesis, we derived tumoroids from the different tumour types. Interestingly, tumoroids derived from inflammation-induced tumours from control mice maintained many of the inflammation-induced DNA hypermethylation alterations and had higher levels of DNA hypermethylation at these regions than tumoroids from DAC-treated mice. Importantly, tumoroids derived from inflammation-induced tumours from the DAC-treated mice proliferated more slowly than those derived from the inflammation-induced tumours from control mice. These studies suggest that inhibition of inflammation-induced DNA hypermethylation may be an effective strategy to reduce inflammation-induced tumorigenesis.

Abstract 5317: JAK2 regulates oxidative damage-induced epigenetic alterations

Abstract Elevated levels of reactive oxygen species at sites of chronic inflammation cause oxidative DNA damage in epithelial cells, contributing to carcinogenesis. Aberrant silencing of key genes by promoter CpG island DNA hypermethylation occurs in many cancers that form at sites of chronic inflammation. Yet, it is not completely understood how these aberrant DNA methylation alterations are initiated. We hypothesize that the recruitment of epigenetic silencing proteins to sites of oxidative DNA damage is important for initiating DNA methylation. Here we demonstrate that after hydrogen peroxide (H2O2) treatment, mismatch repair proteins MSH2 and MSH6 become localized to sites of oxidative DNA damage, resulting in the recruitment of DNA methyltransferase 1 (DNMT1) and Polycomb repressive complex 2 (PRC2) members to chromatin. This recruitment results in the formation of repressive chromatin in the promoters of tumor-suppressor genes and the concomitant transient reduction in their expression. We sought to further understand the mechanism that drives H2O2-induced MSH2 and MSH6 chromatin binding. Janus kinase 2 (JAK2) is a kinase that is canonically known to be activated by cytokines and growth hormones and phosphorylate the transcription factor signal transducer and activator of transcription (STATs). Here, we demonstrate that in response to treatment with H2O2, but not interleukin 6 (IL-6), JAK2 translocates to the nucleus and interacts with MSH2 and MSH6. Nuclear JAK2 is important for H2O2-induced chromatin interaction of MSH2, MSH6, DNMT1 and PRC2 members. Interestingly, H2O2-induces a global and site-specific increase in trimethylation of histone H3 at lysine 27 (H3K27me3) that is also dependent on JAK2. Furthermore, by analyzing TCGA datasets, we demonstrate high JAK2 mRNA expression correlates with CpG island methylator phenotype (CIMP) status in gastric and colorectal cancer. All together, these findings suggest JAK2 plays a key role in initiating oxidative damage-induced epigenetic alterations during inflammation-driven tumorigenesis. Ultimately, understanding the mechanism of initiation of cancer-specific promoter DNA hypermethylation may lead to treatment strategies to reduce aberrant epigenetic changes induced by chronic inflammation and therefore reduce tumorigenesis. Citation Format: Ning Ding, Sam Miller, Heather O'Hagan. JAK2 regulates oxidative damage-induced epigenetic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5317.

Genetic alterations in adult T-cell leukemia/lymphoma.

Adult T‐cell leukemia/lymphoma (ATL) is a peripheral T‐cell neoplasm with a dismal prognosis. It is caused by human T‐cell leukemia virus type‐1 (HTLV‐1) retrovirus. A long latency period from HTLV‐1 infection to ATL onset suggests that not only HTLV‐1 proteins, such as Tax and HBZ, but also additional genetic and/or epigenetic events are required for ATL development. Although many studies have demonstrated the biological functions of viral genes, alterations of cellular genes associated with ATL have not been fully investigated. Recently, a large‐scale integrated genetic analysis revealed the entire landscape of somatic aberrations in ATL. This neoplasm is characterized by frequent gain‐of‐function alterations in components of the T‐cell receptor/NF‐κB signaling pathway, including activating mutations in the PLCG1,PRKCB,CARD11 and VAV1 genes, and CTLA4‐CD28 and ICOS‐CD28 fusions. Importantly, molecules associated with immune surveillance, such as HLA‐A/B,CD58 and FAS, are affected recurrently. Among them, one notable lesion occurs as frequent structural variations that truncate the PD‐L1 3′‐untranslated region, leading to its overexpression. Other genetic targets include transcription factors (IRF4,IKZF2, and GATA3) and chemokine receptors (CCR4,CCR7 and GPR183), which are functionally relevant in normal T cells. A substantial proportion of ATL cases show widespread accumulation of repressive epigenetic changes, such as trimethylation of histone H3 lysine 27 and DNA hypermethylation of CpG islands, which coordinately modulate multiple pathways, including Cys2‐His2 zinc finger genes involved in silencing retroelements. Here we review the current understanding of the genetic/epigenetic aberrations in ATL, focusing on their relevance in its molecular pathogenesis.

Abstract 1002: An epigenetic strategy to degrade the estrogen receptor in breast cancer

Abstract Epigenetic changes in DNA methylation can lead to altered gene expression and the development and progression of breast cancer. Luminal breast tumors are associated with increased DNA hypermethylation of CpG islands while basal, triple negative tumors more commonly have lower levels of DNA methylation. Increasing evidence suggests that aberrant epigenetic alterations in DNA methylation and chromatin structure may also contribute to endocrine resistance in luminal breast cancers. We thus asked whether epigenetic therapy using the demethylating agent, 5-Azacytidine (AZA) is an effective anti-tumorigenic agent in luminal breast cancers, what epigenetic mechanisms might be involved in the response and whether AZA could play a role in the sensitization of tamoxifen resistant cells. We have demonstrated using 26 breast cancer cell lines that luminal B, ER+ breast cancer is more responsive to treatment with AZA than are Her2+ or triple negative (TN) breast cancers. Cells were treated in culture with low nanomolar doses of AZA for 72 hrs, followed by transplantation into mice and weekly palpation of tumors. Tumorgraft measurements show that 67% of luminal B, ER+ cells are sensitive to AZA, whereas only 22% of TN and 25% of Her2+ cells exhibit an anti-tumorigenic response to AZA in mice. Expression data showed that AZA leads to decreases in ER mRNA and protein expression in 50% and 67% of luminal B lines respectively. Further investigation showed that a longer AZA treatment led to lower ERα protein levels and stronger tumor growth inhibition. Protease inhibitor (MG132) treatment rescued ERα protein reduction in 50% of the luminal B cell lines, suggesting that an AZA-mediated, ubiquitin-26S proteasomal pathway may be responsible for the degradation of ER. Gene expression analysis demonstrated that the E1 ubiquitin-activating enzyme, (UBA7-UBE1L) was up-regulated at the mRNA and protein levels by AZA. UBA7 has been suggested to function as a tumor suppressor in lung cancer and elevations in UBA7 have been correlated with longer overall survival in breast cancer. The interaction of UBA7 with ERα protein was validated by immunoprecipitation and over-expression of UBA7 in ZR-75-1 cells led to a reduction in ERα protein. We further analyzed the UBA7 promoter region by Illumina 450K array and bisulfite sequencing in our cell lines and identified two CpG rich sequences in the promoter that may be important for AZA induced UBA7 promoter demethylation and gene re-expression. We also demonstrated in both tamoxifen sensitive and resistant cells that combination therapy with both AZA and tamoxifen was more effective at inhibiting tumor growth than the single agents. Studies are ongoing in our lab to better understand this response. Our data suggest that AZA may be clinically useful to inhibit the growth of some luminal breast tumors via its actions to increase expression of UBA7 and to degrade ER and may have efficacy in combination with anti-estrogen treatments. Citation Format: Huili Li, Foteinos-Ioannis Dimitrakopoulos, Meredith Stone, Lauren Murphy, Stephen Baylin, Cynthia A. Zahnow. An epigenetic strategy to degrade the estrogen receptor in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1002. doi:10.1158/1538-7445.AM2017-1002

The mechanism of apoliprotein A1 down-regulated by Hepatitis B virus.

BackgroundHepatitis B virus (HBV) infection correlated with the development of cirrhosis, liver failure and hepatocellular carcinoma (HCC), poses a huge health burden on the global community. However, the pathogenesis of chronic hepatitis B (CHB) remains unclear. Apolipoprotein A1 (ApoA1) mainly secreted by hepatocytes, represents the major protein component of high-density lipoprotein. ApoA1 secretion may be disrupted by HBV infection. In this study, we mainly investigated the molecular mechanism of ApoA1 down regulated by HBV for revealing the pathogenesis of CHB.MethodsApoA1 expression in livers of CHB patients as well as healthy controls were performed by Real-time PCR (RT-PCR) and Western blot. The serum ApoA1 levels were measured by Enzymed-linked immunosorbent assay (ELISA). Expression of ApoA1 mRNA and protein levels were performed by RT-PCR and Western blot in human hepatoma HepG2 cells and subline HepG2.2.15 cells. HBV expression construct, pHBV1.3 were transfected into HepG2, the changes of ApoA1 mRNA and protein expression were detected by RT-PCR and Western blot. To further study the mechanism of ApoA1 down regulation by HBV, 11 CpG islands in ApoA1 promotor were tested for DNA methylation status by MSP. HepG2.2.15 cell lines were treated with DNA methyltransferase inhibitor 5-aza-deoxycytidine (5-aza-dC), then, expression of ApoA1 mRNA and HBV particles in the supernatant, as well as ApoA1 protein levels were detected by RT-PCR and Western blot. Secretion of HBsAg and HBeAg in HepG2 cells cotransfected with pApoA1 and pHBV1.3 constructs was tested by ELISA. Meanwhile, secretion of HBsAg and HBeAg in the supernatant were quantified by ELISA in the HepG2.2.15 cells treated with 5-aza-dC plus ApoA1 siRNA.ResultsExpression of ApoA1 mRNA and protein levels, as well as serum ApoA1 levels in CHB patients were decreased corresponding healthy controls in vivo. In addition, the expression of ApoA1 mRNA and protein levels were down regulated in HepG2.2.15 cells correponding HepG2 cells, 11 CpG islands in ApoA1 promoter were tested for methylation status by MSP in HepG2.2.15 cells compared to HepG2 cells, while two CpG islands were found hypermethylated. Expression of ApoA1 mRNA and protein levels were increased in HepG2.2.15 cells treated with DNA methyltransferase inhibitor 5-aza-dC. Furthermore, overexpression of ApoA1 can enhance HBV expression in HepG2 cells while the inhibitory effect of 5-aza-dC on HBV expression was completely abolished by blocking 5-aza-dC-induced up-regulation of ApoA1 using RNAi.ConclusionsEpigenetic silencing of ApoA1 gene expression by CpG island DNA hypermethylation induced by HBV may contribute to the pathogenesis of CHB.

Abstract 1049: Prognostication of patients with clear cell renal cell carcinomas based on quantification of DNA methylation levels of CpG island methylator phenotype marker genes using anion exchange high performance liquid chromatography

Abstract Background: We have identified CpG island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (ccRCCs) characterized by accumulation of DNA hypermethylation of CpG islands, tumor aggressiveness and poorer patient outcome. Furthermore, the criteria for prognostication of patients with ccRCCs have been established based on DNA methylation levels of ccRCC-specific CIMP marker genes. Recently, we have developed an anion exchange High Performance Liquid Chromatography (HPLC) column for detection of methylated DNA. To make the prognostication of patients with ccRCCs clinically applicable, the newly developed HPLC column has been applied to DNA methylation analysis of CIMP marker genes. Methods: Genomic DNA extracted from tumorous tissue samples of nephrectomy specimens of 5 patients with CIMP-positive ccRCCs and 5 patients with CIMP-negative ccRCCs was amplified by Polymerase Chain Reaction (PCR) after sodium bisulfite conversion with the EpiTect Bisulfite Kit (QIAGEN). Three-hundred and eighty-four bp-PCR products encompassing the promoter region of the FAM150A gene, a CIMP marker gene, and including 39 CpG sites were subjected to the HPLC column. DNA methylation levels were quantitatively evaluated using 0% and 100% methylated DNA controls (QIAGEN). Results: The analysis time for all samples was within 10 minutes. The retention time difference between 0% and 100% methylated DNA controls was 0.34 min. CIMP-negative ccRCCs were detected as a distinct single peak which had similar retention time with the 0% methylated DNA control, whereas CIMP-positive ccRCCs showed a bimodal peak pattern. Retention time of the later peak of CIMP-positive ccRCCs was similar with the 0% methylated DNA control. On the other hand, an early peak was eluted at various positions depending on its DNA methylation level. Based on the differences of chromatogram patterns, CIMP-positive ccRCCs were completely discriminated from CIMP-negative ccRCCs. DNA methylation levels of the FAM150A gene of each tissue sample calculated from retention time were consistent with those previously evaluated using MassARRAY system (Sequenom). Conclusion: Using newly developed anion exchange HPLC column, tissue specimens of CIMP-positive ccRCCs were readily, quickly and clearly distinguished from those of CIMP-negative ccRCCs, indicating that this HPLC method may be suitable for clinical application of prognostication of patients with ccRCCs based on CIMP diagnosis. Citation Format: Takuya Yotani, Yuriko Yamada, Eri Arai, Ying Tian, Masahiro Gotoh, Motokiyo Komiyama, Hiroyuki Fujimoto, Michiie Sakamoto, Yae Kanai. Prognostication of patients with clear cell renal cell carcinomas based on quantification of DNA methylation levels of CpG island methylator phenotype marker genes using anion exchange high performance liquid chromatography. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1049. doi:10.1158/1538-7445.AM2015-1049

Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas.

CpG‐island methylator phenotype (CIMP)‐positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP‐positive renal carcinogenesis. Genome (whole‐exome and copy number), transcriptome and proteome (two‐dimensional image converted analysis of liquid chromatography‐mass spectrometry) analyses were performed using tissue specimens of 87 CIMP‐negative and 14 CIMP‐positive clear cell RCCs and corresponding specimens of non‐cancerous renal cortex. Genes encoding microtubule‐associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non‐synonymous single‐nucleotide mutations and insertions/deletions) in CIMP‐positive RCCs, whereas CIMP‐negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP‐positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the “The metaphase checkpoint (p = 1.427 × 10−6),” “Role of Anaphase Promoting Complex in cell cycle regulation (p = 7.444 × 10−6)” and “Spindle assembly and chromosome separation (p = 9.260 × 10−6)” pathways. Quantitative RT‐PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP‐positive than in CIMP‐negative RCCs. All CIMP‐positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP‐positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP‐positive RCCs.

Abstract 18405: Epidrug Treatment Rescues Proliferation and Differentiation in Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients: A Case of Epimetabolic Memory

Introduction: The origin of metabolic/epigenetic memory remains elusive. It, however, may be at the basis of chronic diseases and functional alteration associated to ageing and cardiovascular disease. HYPOTHESIS: Diabetes is known to alter cellular metabolism causing defects in tissue/organ regeneration. In this study we asked whether human cardiac stromal cells from diabetic and normoglycaemic voluntary donors revealed differences associated to their original metabolism and whether the alterations could be rescued by epigenetically active drugs. METHODS and RESULTS: D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of histone H3 lysine 9 and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5- related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-a general control of amino acid synthesis (GCN5a), determining a relative decreasein total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. CONCLUSIONS: This study investigated the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Abstract 2316: Multilayer omics analyses in CpG island methylator phenotype clear cell renal cell carcinomas

Abstract [Background] We have reported that CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poorer patient outcome. We have established the criteria for CIMP diagnosis in clear cell RCCs by quantification of DNA methylation levels of RCC-specific CIMP marker genes. [Purpose] The aim of this study was to reveal the molecular pathways which significantly participate in CIMP-positive renal carcinogenesis and become a therapeutic target of CIMP-positive clear cell RCCs. [Methods] Our previous methylome analysis using Illumina Infinium HumanMethylation27 BeadArray identified CIMP-negative and -positive clear cell RCCs in the present cohort (n=101). In the present study, genome (whole-exome), transcriptome and proteome analyses using Agilent SureSelect All Exon capture followed by sequencing on an Illumina HiSeq 2000 platform, Agilent SurePrint Human Gene Expression microarray and two dimensional image converted analysis of liquid chromatography-mass spectrometry, respectively, were performed in tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and the corresponding non-cancerous renal cortex tissue specimens. [Results] The number of genes showing genetic aberrations in CIMP-negative clear cell RCCs was quite low, indicating that CIMP-negative RCCs lacked distinct genetic characteristics. On the other hand, many genes, such as genes encoding hisotone modification enzymes and tumor-suppressor genes, showed 10% or more incidence of genetic aberrations in CIMP-positive clear cell RCCs. Based on MetaCore pathway analysis, although mRNA or protein expression alterations in CIMP-negative clear cell RCCs were not significantly accumulated in any molecular pathways, such alterations in CIMP-positive clear cell RCCs were accumulated in the 19 pathways (P&amp;lt;0.05), such as molecular pathways involved in cell cycle and cell adhesion. Quantitative RT-PCR analysis verified expression alterations of genes involved in the most frequently affected pathway in CIMP-positive clear cell RCCs. [Conclusion] The most frequently affected pathway may be a novel therapeutic target of CIMP-positive clear cell RCCs. We are now examining the effectiveness of the inhibitor of the most frequently affected pathway in RCC cell lines showing CIMP. Citation Format: Eri Arai, Hiromi Sakamoto, Masaya Ono, Yoriko Takahashi, Sayaka Miyata, Hiroyuki Fujimoto, Masahiro Gotoh, Tesshi Yamada, Yae Kanai. Multilayer omics analyses in CpG island methylator phenotype clear cell renal cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2014-2316

The Histone Acetylase Activator Pentadecylidenemalonate 1b Rescues Proliferation and Differentiation in the Human Cardiac Mesenchymal Cells of Type 2 Diabetic Patients

This study investigates the diabetes-associated alterations present in cardiac mesenchymal cells (CMSC) obtained from normoglycemic (ND-CMSC) and type 2 diabetic patients (D-CMSC), identifying the histone acetylase (HAT) activator pentadecylidenemalonate 1b (SPV106) as a potential pharmacological intervention to restore cellular function. D-CMSC were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence. A global histone code profiling of D-CMSC revealed that acetylation on histone H3 lysine 9 (H3K9Ac) and lysine 14 (H3K14Ac) was decreased, whereas the trimethylation of H3K9Ac and lysine 27 significantly increased. These observations were paralleled by a downregulation of the GCN5-related N-acetyltransferases (GNAT) p300/CBP-associated factor and its isoform 5-α general control of amino acid synthesis (GCN5a), determining a relative decrease in total HAT activity. DNA CpG island hypermethylation was detected at promoters of genes involved in cell growth control and genomic stability. Remarkably, treatment with the GNAT proactivator SPV106 restored normal levels of H3K9Ac and H3K14Ac, reduced DNA CpG hypermethylation, and recovered D-CMSC proliferation and differentiation. These results suggest that epigenetic interventions may reverse alterations in human CMSC obtained from diabetic patients.

Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53- and DNMT1(DNA methyltransferase 1)-independent manner

Epigenetic silencing of gene expression is important in cancer. Aberrant DNA CpG island hypermethylation and histone modifications are involved in the aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) is a H3K4 (histone H3 Lys4) demethylase associated with gene repression and is overexpressed in multiple cancer types. LSD1 has also been implicated in targeting p53 and DNMT1 (DNA methyltransferase 1), with data suggesting that the demethylating activity of LSD1 on these proteins is necessary for their stabilization. To examine the role of LSD1 we generated LSD1 heterozygous (LSD1+/−) and homozygous (LSD1−/−) knockouts in the human colorectal cancer cell line HCT116. The deletion of LSD1 led to a reduced cell proliferation both in vitro and in vivo. Surprisingly, the knockout of LSD1 in HCT116 cells did not result in global increases in its histone substrate H3K4me2 (dimethyl-H3K4) or changes in the stability or function of p53 or DNMT1. However, there was a significant difference in gene expression between cells containing LSD1 and those null for LSD1. The results of the present study suggested that LSD1 is critical in the regulation of cell proliferation, but also indicated that LSD1 is not an absolute requirement for the stabilization of either p53 or DNMT1.