CpG Hypermethylation Research Articles

Abstract A019: Chimeric antigen receptors (CARs) as a low-impact treatment of pediatric ependymomas

Abstract Ependymoma is the third most common paediatric brain tumor and can occur anywhere along the neuroaxis, the most common location being the posterior fossa (PF). Surgery followed by radiation therapy (>4 years of age) is to date the most effective treatment regimen; chemotherapy is not part of the current standard of care as multiple clinical trials have failed to show any survival benefits. Despite extensive characterization of ependymoma, few credible oncogenes and tumor suppressor genes have been identified, in fact, up to 50% of PF ependymoma cases exhibit a balanced and bland genomic profile. Consequently, actionable mutations are not currently identifiable in the vast majority of cases. We are investigating candidate chimeric antigen receptor (CAR)- strategies to treat posterior fossa-A ependymoma (PFA) — a subtype that has the worst associated prognosis and is found predominately in infants. Probing of our extensive ependymoma gene expression dataset has identified several potential CAR target antigens, including HER2, EPHA2 and IL13Rα2, these have been further verified with tissue microarrays (TMAs). Using our orthotopic xenograft models of PFA ependymoma, we are confident from our preliminary analysis that HER2-positive PFA ependymomas respond to HER2-specific CAR treatment. Furthermore, the use of a TRI-CAR, tailored to target cells expressing EPHA2, IL13Rα2 and HER2, both collectively and independently, increases the efficacy and specificity as a therapy for both primary and recurrent ependymoma. To ensure we are using the most effective negative controls when confirming the therapeutic response of our CARs, we are silencing our genes of interest in our patient-derived ependymoma lines using shRNA and CRISPR-technology for HER2, EPHA2 and IL13Rα2. Whole-genome and whole-exome sequencing has demonstrated, that in comparison to PFB ependymomas, PFA tumors have more methylated CpG sites, and more genes that are transcriptionally silenced by CpG hypermethylation. To reduce this silencing and potentially improve therapeutic response to our CARs, we are using Azacitadine in combination with our CAR therapies. In parallel with our pre-clinical trials, we are performing safety trials to assess the administration of intravenous versus intrathecal HER2 CAR delivery. Patients presenting with a brain tumor frequently present with a leaky blood-CSF and blood-Brain barrier, therefore we aim to observe whether CARs injected intrathecally can be found within the blood and vice versa. Using a HER2 Copy Number Assay, we observed two significant results: firstly, HER2 CARs injected intrathecally, in the presence of a HER2-positive brain tumor, remain within the central nervous and are not found within the blood; secondly, HER2 CARs administered intraventrically in the presence of a HER2-positive brain tumor, migrate to the CSF (P = 0.009). Results from this project have the potential to create a paradigm shift in our approach to the treatment of ependymoma, including the initiation of a clinical trial for children with PFA ependymoma. This technology has the real potential to improve cure rates whilst also dramatically improving the quality of life for surviving patients by reducing the impact that current treatments have on normal brain development. Citation Format: Laura K. Donovan, Kevin J. Bielamowicz, Alex Manno, Nabil Ahmed, Michael D. Taylor. Chimeric antigen receptors (CARs) as a low-impact treatment of pediatric ependymomas [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A019.

PPARβ/δ activation protects against corticosterone-induced ER stress in astrocytes by inhibiting the CpG hypermethylation of microRNA-181a

Increasing evidence indicates that peroxisome proliferator-activated receptors (PPARs) play neuroprotective roles in various neurodegenerative disease models in vivo and in vitro. However, the underlying mechanisms remain unclear. Astrocyte proliferation is a key process in neural development and plays significant roles in the regeneration of neural tissue after a penetrating injury. Corticosterone can significantly reduce the expression of glial fibrillary acid protein (GFAP) in cultured rat hippocampal astrocytes in vitro, and induce astrocytic dysfunction. Our research found that corticosterone treatment resulted in astrocyte damage and reduced the expression of PPARβ/δ. GW0742, a selective and high-affinity PPARβ/δ agonist, attenuated the corticosterone-induced astrocyte damage, but also significantly reversed the increase in the expression of GRP78 and CHOP, the two predominant proteins in endoplasmic reticulum (ER) stress. Moreover, GW0742 decreased the levels of caspase-12 and cleaved caspase-3, thereby protecting astrocytes against corticosterone-induced astrocyte apoptosis. We then confirmed that GRP78 was a target gene of microRNA-181a and found that PPARβ/δ activation increased microRNA-181a levels. Finally, we demonstrated that PPARβ/δ activation by GW0742 noticeably inhibited the activities and expression of DNA methyltransferases, and reduced the corticosterone-induced CpG island hypermethylation of microRNA-181a1 in astrocytes. Therefore, the present study is the first to reveal that PPARβ/δ activation suppresses CpG island hypermethylation-associated silencing of microRNA-181a and thereby protects against ER stress-induced damage in astrocytes. Our findings suggest that PPARβ/δ activation in astrocytes might be a promising target for regulating ER stress-induced astrocytic injury.

Possible role of intragenic DNA hypermethylation in gene silencing of the tumor suppressor gene NR4A3 in acute myeloid leukemia

Expression of the tumor suppressor gene NR4A3 is silenced in the blasts of acute myeloid leukemia (AML), irrespective of the karyotype. Although the transcriptional reactivation of NR4A3 is considered to have a broad-spectrum anti-leukemic effect, the therapeutic modalities targeting this gene have been hindered by our minimal understanding of the transcriptional mechanisms regulating its expression, particularly in human AML. Here we show the role of intragenic DNA hypermethylation in reducing the expression of NR4A3 in AML. Bisulfite sequencing analysis revealed that CpG sites at the intragenic region encompassing exon 3 of NR4A3, but not the promoter region, are hypermethylated in AML cell lines and primary AML cells. A DNA methyltransferase inhibitor restored the expression of NR4A3 following a reduction in DNA methylation levels at intragenic CpG sites. The in silico data revealed an enrichment of H3K4me1 and H2A.Z at exon 3 of NR4A3 in human non-malignant cells but that was excluded specifically in leukemia cells with CpG hypermethylation. This suggests that exon 3 represents a functional regulatory element involved in the transcriptional regulation of NR4A3. Our findings improve the current understanding of the mechanism underlying NR4A3 silencing and facilitate the development of NR4A3-targeted therapy.

Folate deprivation induces cell cycle arrest at G0/G1 phase and apoptosis in hippocampal neuron cells through down-regulation of IGF-1 signaling pathway

Folate deficiency contributes to impaired adult hippocampal neurogenesis, yet the mechanisms remain unclear. Here we use HT-22 hippocampal neuron cells as model to investigate the effect of folate deprivation (FD) on cell proliferation and apoptosis, and to elucidate the underlying mechanism. FD caused cell cycle arrest at G0/G1 phase and increased the rate of apoptosis, which was associated with disrupted expression of folate transport and methyl transfer genes. FOLR1 and SLC46A1 were (P<0.01) down-regulated, while SLC19A1 was up-regulated (P<0.01) in FD group. FD cells exhibited significantly (P<0.05) higher protein content of BHMT, MAT2b and DNMT3a, as well as increased SAM/SAH concentrations and global DNA hypermethylation. The expression of the total and all the 3 classes of IGF-1 mRNA variants was significantly (P<0.01) down-regulated and IGF-1 concentration was decreased (P<0.05) in the culture media. IGF-1 signaling pathway was also compromised with diminished activation (P<0.05) of STAT3, AKT and mTOR. CpG hypermethylation was detected in the promoter regions of IGF-1 and FOLR1 genes, while higher SLC19A1 mRNA corresponded to hypomethylation of its promoter. IGF-1 supplementation in FD media significantly abolished FD-induced decrease in cell viability. However, IGF-1 had limited effect in rescuing the cell phenotype when added 24h after FD. Taken together, down-regulation of IGF-1 expression and signaling is involved in FD-induced cell cycle arrest and apoptosis in HT-22 hippocampal neuron cells, which is associated with an abnormal activation of methyl transfer pathway and hypermethylation of IGF-1 gene promoter.

Curcumin attenuates cyclosporine A‑induced renal fibrosis by inhibiting hypermethylation of the klotho promoter.

Chronic kidney disease is increasingly considered to be a worldwide public health problem and usually leads to renal fibrosis. In the present study, curcumin, a polyphenol pigment extracted from turmeric, was demonstrated to exert protective effects on renal fibrosis via the suppression of transforming growth factor‑β (TGF‑β) downstream signaling, such as plasminogen activator inhibitor‑1 (PAI‑1), α‑smooth muscle actin (α‑SMA) and collagenI (ColI) downregulation. The present findings demonstrate that curcumin exerted a protective effect on cyclosporineA‑induced renal fibrosis via a klotho (KL)‑dependent mechanism, which inhibits the TGF‑β signaling pathway. Further research indicated that curcumin induced KL expression in HK‑2 tubular epithelial cells by inhibiting CpG hypermethylation in the KL promoter, which mediates the loss of expression in cells. Methylation‑specific polymerase chain reaction (PCR) combined with bisulfite sequencing identified numerous key CpG sites, such as 249, 240 and 236, whose methylation statuses are important for KL expression. A PCR reporter assay was utilized to further confirm these findings. In addition, the effects of curcumin on the regulation of DNA methyltransferase1 (Dnmt1) expression were evaluated, and the data suggest that curcumin inhibits Dnmt1 expression and restricts CpG hypermethylation. Thus, the current study reveals that curcumin attenuated renal fibrosis by suppressing CpG methylation in the KL promoter, thus inducing KL expression, which inhibited TGF‑β signaling, which may provide a novel therapeutic approach for the treatment of renal fibrosis.

Knockdown of Plakophilin 2 Downregulates miR-184 Through CpG Hypermethylation and Suppression of the E2F1 Pathway and Leads to Enhanced Adipogenesis In Vitro.

PKP2, encoding plakophilin 2 (PKP2), is the most common causal gene for arrhythmogenic cardiomyopathy. To characterize miRNA expression profile in PKP2-deficient cells. Control and PKP2-knockdown HL-1 (HL-1(Pkp2-shRNA)) cells were screened for 750 miRNAs using low-density microfluidic panels. Fifty-nine miRNAs were differentially expressed. MiR-184 was the most downregulated miRNA. Expression of miR-184 in the heart and cardiac myocyte was developmentally downregulated and was low in mature myocytes. MicroRNA-184 was predominantly expressed in cardiac mesenchymal progenitor cells. Knockdown of Pkp2 in cardiac mesenchymal progenitor cells also reduced miR-184 levels. Expression of miR-184 was transcriptionally regulated by the E2F1 pathway, which was suppressed in PKP2-deficient cells. Activation of E2F1, on overexpression of its activator CCND1 (cyclin D1) or knockdown of its inhibitor retinoblastoma 1, partially rescued miR-184 levels. In addition, DNA methyltransferase-1 was recruited to the promoter region of miR-184, and the CpG sites at the upstream region of miR-184 were hypermethylated. Treatment with 5-aza-2'-deoxycytidine, a demethylation agent, and knockdown of DNA methyltransferase-1 partially rescued miR-184 level. Pathway analysis of paired miR-184:mRNA targets identified cell proliferation, differentiation, and death as the main affected biological processes. Knockdown of miR-184 in HL-1 cells and mesenchymal progenitor cells induced and, conversely, its overexpression attenuated adipogenesis. PKP2 deficiency leads to suppression of the E2F1 pathway and hypermethylation of the CpG sites at miR-184 promoter, resulting in downregulation of miR-184 levels. Suppression of miR-184 enhances and its activation attenuates adipogenesis in vitro. Thus, miR-184 contributes to the pathogenesis of adipogenesis in PKP2-deficient cells.

Abstract 1095: S100A4 is post-transcriptionally inhibited by miR-505-5p and miR-520c-3p in colorectal cancer

Abstract The calcium binding protein S100A4 induces epithelial mesenchymal transition (EMT), migration, invasion, angiogenesis and metastasis. Its induced expression is reported in several cancer types and correlates with poor prognosis. S100A4 is transcriptionally regulated by Sp1, AP1 and CCAAT/enhancer binding protein β transcription factors and its regulation epigenetically controlled by CpG hyper methylation. However, apart from S100A4 functional and transcriptional regulatory aspects, post-transcriptional regulation is not yet elucidated. In this study, we show that specific microRNAs (miR) target the 3’UTR of S100A4. Luciferase-reporter assays with wild-type and mutated miR-505-5p and miR-520c-3p seed sequences of S100A4-3’UTR confirmed the specificity of targeting. Similarly, overexpression of these miRs reduced the S100A4 and inhibition of these miRs function increased the S100A4 protein significantly. Moreover, co-transfection of either S100A4 expressing vector or siRNA-S100A4 along with the miR-505-5p and miR-520c-3p significantly altered the S100A4 mediated migration and invasion in colorectal cancer (CRC) cell lines (p = 0.05). Both these miRs were down-regulated in CRC cell lines and 5 Aza treatment increased these miRs expression and reduced the S100A4 protein amounts. Moreover, in a panel of CRC resected patient tissues (n = 59), miR-520c-3p significantly down regulated (p = 0.03). miR-520c (located in Chr 19, in a big cluster) upstream region was checked for methylation status using methylation specific PCR and combined bisulfite restriction analysis (COBRA) in CRC cell lines, metastatic and non-metastatic CRC resected tissues. We found the upstream promoter region hyper methylated irrespective of metastasis status of patients and in all CRC cell lines checked. 5 Aza treatment of S100A4 less or no expressing cells line Colo206 and Colo320 showed induced miR-520c expression and reduced S100A4 protein in Colo320. Colo206 cells did not show any alternations in expression of S100A4. Taken together, our findings demonstrate that S100A4 is post-transcriptionally regulated by tumor suppressor genes miR-505c and miR-520c and especially miR-520c expression is epigenetically silenced in CRC. Citation Format: Giridhar Mudduluru, Katharina Ilm, Steffen Fuchs, Ulrike Stein. S100A4 is post-transcriptionally inhibited by miR-505-5p and miR-520c-3p in colorectal cancer. [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 1095.

Abstract 3664: RARRES2 functions as a direct tumor suppressor by promoting beta-catenin phosphorylation and degradation independent of CMKLR1 and CMKLR1-mediated antitumor immune response in adrenocortical carcinoma

Abstract Tumor suppressors and the immune system are critical players in tumor suppression. However, little is known about whether a tumor suppressor can function through both immune-dependent and immune-independent mechanisms. RARRES2 is a small secreted protein that is down-regulated in multiple human cancer types such as adrenocortical carcinoma (ACC), hepatocellular carcinoma (HCC) and melanoma. It has been shown that RARRES2 can play an indirect tumor-suppressive role by acting as a chemoattractant to recruit anti-cancer immune cells expressing its receptor CMKLR1 to sites of tumor. In this study, we show that epigenetic CpG hypermethylation of RARRES2 in vivo and in vitro in ACC causes RARRES2 down-regulation. We also found that RARRES2 can function as a direct tumor suppressor and that its tumor suppressive effect does not depend on the recruitment of CMKLR1-expressing immune cells. We show that although exogenous treatment of cultured cells with recombinant human RARRES2 protein did not show any tumor-suppressive effect, transient overexpression of RARRES2 significantly inhibited cellular invasion. Stable overexpression of RARRES2 in ACC cell line NCI-H295R led to significantly reduced cell proliferation, cell invasion, and tumorigenecity in a RARRES2 dose-dependent manner. Using xenografts of stable NCI-H295R cell lines in two immunodeficient mouse models (athymic nude mice and NSG mice), we found that in the absence of functional immune cells, stable RARRES2 overexpression can still significantly suppress tumor growth in vivo. RARRES2 overexpression promoted beta-catenin phosphorylation and degradation, reduced the phosphorylation of p38 MAP kinase and inhibited the secretion of matrix metalloprotease protein 10 (MMP10) from cancer cells in a RARRES2 dose-dependent manner. Taken into account that CMKLR1 expression is barely detectable in all the cell lines tested, our results present the first evidence that RARRES2 can function as a direct tumor suppressor independent of CMKLR1 and CMKLR1-mediated anti-tumor immune response. Citation Format: Yi Liu-Chittenden, Kelli Gaskins, Sophie Wang, Electron Kebebew. RARRES2 functions as a direct tumor suppressor by promoting beta-catenin phosphorylation and degradation independent of CMKLR1 and CMKLR1-mediated antitumor immune response in adrenocortical 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 3664.

Independent Tumor Origin in Two Cases of Synchronous Bilateral Clear Cell Renal Cell Carcinoma.

Bilateral renal cell carcinomas (RCCs) pose a challenge for clinical treatment and management. Most bilateral RCCs are sporadic, and do not show a hereditary pattern indicative of VHL syndrome or other inherited cancers. The origin and evolution of these sporadic bilateral RCCs remains elusive. We obtained normal and tumor samples from two male patients suffering from early stage synchronous bilateral clear cell RCC (ccRCC), and analyzed genomic DNA using whole exome sequencing and bisulfite pyrosequencing. We detected distinct 3p loss of heterozygosity (LOH) in both tumors in each patient. Two tumors within the same patient harbored distinct driver mutations and different CpG hypermethylation sites in the VHL promoter. Moreover, tumors exhibit independent evolutionary trajectories. Therefore, distinct 3p LOH, combined with contingent driver gene mutations and independent VHL hypermethylation, led to independent tumor origin and parallel evolution of bilateral ccRCC in these two patients. Our results indicate that tumors in these two cases were not due to common germline oncogenic mutations. They were results of multiple de novo mutations in each kidney, rather than primary ccRCC with contralateral renal metastasis. Therefore, histopathologic and genetic profiling from single tumor specimen may underestimate the mutational burden and somatic heterogeneity of bilateral ccRCCs.

Gene and transposable element methylation in great tit (Parus major) brain and blood.

BackgroundStudies on vertebrate DNA methylomes have revealed a regulatory role of tissue specific DNA methylation in relation to gene expression. However, it is not well known how tissue-specific methylation varies between different functional and structural components of genes and genomes. Using whole-genome bisulfite sequencing data we here describe both CpG and non-CpG methylation profiles of whole blood and brain tissue in relation to gene features, CpG-islands (CGIs), transposable elements (TE), and their functional roles in an ecological model species, the great tit (Parus major).ResultsWe show that hypomethylation at the transcription start site (TSS) is enriched in genes with functional classes that relate directly to processes specific to each tissue type. We find that 6877 (~21 %) of the CGIs are differentially methylated between blood and brain, of which 1186 and 2055 are annotated to promoter and intragenic regions, respectively. We observe that CGI methylation in promoter regions is more conserved between tissues compared to CGI methylation in intra and inter-genic regions. Differentially methylated CGIs in promoter and intragenic regions are overrepresented in genomic loci linked to development, suggesting a distinct role for CGI methylation in regulating expression during development. Additionally, we find significant non-CpG methylation in brain but not in blood with a strong preference for methylation at CpA dinucleotide sites. Finally, CpG hypermethylation of TEs is significantly stronger in brain compared to blood, but does not correlate with TE activity. Surprisingly, TEs showed significant hypomethylation in non-CpG contexts which was negatively correlated with TE expression.ConclusionThe discovery that TSS methylation levels are directly linked to functional classes related to each tissue provides new insights in the regulatory role of DNA-methylation patterns. The dominant sequence motifs for brain non-CpG methylation, similar to those found in mammals, suggests that a conserved non-CpG regulatory mechanism was already present in the amniote ancestor. The negative correlation between brain non-CpG methylation and TE activity (not found for CpG methylation) suggests that non-CpG is the dominant regulatory form of methylation in TE silencing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2653-y) contains supplementary material, which is available to authorized users.

Aberrant expression of the candidate tumor suppressor gene DAL-1 due to hypermethylation in gastric cancer.

By allelotyping for loss of heterozygosity (LOH), we previously identified a deletion region that harbors the candidate tumor suppressor gene DAL-1 at 18p11.3 in sporadic gastric cancers (GCs). The expression and function of DAL-1 in GCs remained unclear. Here, we demonstrated that the absence of or notable decreases in the expression of DAL-1 mRNA and protein was highly correlated with CpG hypermethylation of the DAL-1 promoter in primary GC tissues and in GC cell lines. Furthermore, abnormal DAL-1 subcellular localization was also observed in GC cells. Exogenous DAL-1 effectively inhibited cancer cell proliferation, migration, invasion and epithelial to mesenchymal transition (EMT); exogenous DAL-1 also promoted apoptosis in GC AGS cells. When endogenous DAL-1 was knocked down in GC HGC-27 cells, the cells appeared highly aggressive. Taken together, these findings provide solid evidence that aberrant expression of DAL-1 by hypermethylation in the promoter region results in tumor suppressor gene behavior that plays important roles in the malignancy of GCs. Understanding the role of it played in the molecular pathogenesis of GC, DAL-1 might be a potential biomarker for molecular diagnosis and evaluation of the GC.

Transcriptional Suppression of Renal Antioxidant Enzyme Systems in Guinea Pigs Exposed to Polymerized Cell-Free Hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and plasma volume-expanding therapeutics though their potential to promote oxidative tissue injury has raised safety concerns. Using a guinea pig exchange transfusion model, we examined the effects of polymerized bovine hemoglobin (HbG) on the transcriptional regulation, activity, and expression of the renal antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). HbG infusion downregulated the mRNA levels for genes encoding SOD isoforms 1-3, GPx1, GPx3, GPx4, and CAT. This transcriptional suppression correlated with decreased enzymatic activities for SOD, CAT, and GPx. Immunostaining revealed decreased protein expression of SOD1, CAT, and GPx1 primarily in renal cortical tubules. DNA methylation analyses identified CpG hypermethylation in the gene promoters for SOD1-3, GPx1, GPx3, and GPx4, suggesting an epigenetic-based mechanism underlying the observed gene repression. HbG also induced oxidative stress as evidenced by increased renal lipid peroxidation end-products and 4-HNE immunostaining, which could be the result of the depleted antioxidant defenses and/or serve as a trigger for increased DNA methylation. Together, these findings provide evidence that the renal exposure to HbG suppresses the function of major antioxidant defense systems which may have relevant implications for understanding the safety of hemoglobin-based products.

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression throughFxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells

Background: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention.Objective: We investigated howAPC inactivation influences the regulation ofFXR expression in colonic mucosal cells. We hypothesized thatAPC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation.Methods: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J andApc-deficient (ApcMin/+) male mice, respectively. The expression ofFxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation ofFxr in bisulfonated genomic DNA. In vitro, we measured the impact ofAPC inactivation and deoxycholic acid (DCA) treatment onFXR expression in human colon cancer HCT-116 cells transfected with silencing RNA forAPC and HT-29 cells carrying inactivatedAPC.Results: InApcMin/+ mice, constitutive CpG methylation of theFxrα3/4 promoter was linked to reduced (60–90%) baselineFxr,Ibabp, andShp and increasedCox-2 expression in apparently normal adjacent mucosa and colon tumors.Apc knockdown in HCT-116 cells increased cellular myelocytomatosis(c-MYC) and lowered (∼50%)FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA inducedFXR expression and lowered CpG methylation ofFXR.Conclusions: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.

Abstract A52: Gene alterations associated with clinical characteristics of bladder cancer

Abstract Next-generation sequencing (NGS) of urologic tumor genomes has identified frequently altered cancer genes. We recently examined the exomes of bladder tumors (BCa) from 54 U.S. patients and 99 Chinese patients to identify novel, altered cancer genes associated with clinical characteristics of disease. Exome capture of DNA from tumor and normal urothelial tissue using probes for ~180,000 protein-coding exons and microRNA loci; DNA fragment libraries were sequenced on a HiSeq 2000 platform (Illumina); and base calls on paired-end, 100 bp reads were generated using the Genome Analyzer Pipeline, v. 1.3 and standard parameters. Gene expression was analyzed using quantitative PCR, telomere length by a standard assay, and KDM6A function using cell-based assays in T24T BCa cells. Copy number variation (CNV), aneuploidy, and CpG methylation were analyzed as recently described. Telomerase (TERT) was the most frequently altered by somatic sequence changes in 37/54 (69%) of U.S. patient tumors. The TERT promoter was altered by frequent germline nucleotide changes in 30/54 (56%) tumors. Most of the TERT germline variants were novel (19/20) and three variants were confirmed in distinct tumors as either somatic or germline, including the most frequent variant, c. –245 T&amp;gt;C (rs2853669). The somatic TERT promoter alterations did not correlate with other somatic BCa gene alterations, but were associated with increased TERT expression and with significantly shorter telomeres in tumors as compared to matched normal tissue. The stromal antigen 2 (STAG2) gene was the most frequently altered novel BCa gene in Chinese patients, by somatic, deleterious sequence changes in 11% of tumors; CNV deletions (5%); and promoter CpG hypermethylation leading to presumed expression silencing (7/30, 23%). STAG2 encodes a component of the cohesion complex involved in sister chromatid cohesion and segregation. STAG2 mutations were associated with an increased number of chromosomal arm copy number changes corresponding to higher tumor cell aneuploidy, and mutations were associated with a significantly reduced patient survival by Kaplan-Meier survival analysis. In total, 32/99 (32%) of tumors displayed SCCS pathway gene alterations. The most frequently altered epigenetic gene was the histone lysine-specific demethylase 6A (KDM6A/UTX) in 24% of U.S. and 30% of Chinese patient tumors. Experimentally, we show KDM6A loss in human BCa T24T cells enhanced in vitro proliferation and cell migration, and in vivo tumor growth in mice. Re-expression reversed these effects, confirming KDM6A loss drives the BCa phenotype. Somatic KDM6A alterations were associated with somatic BRCA1-associated protein-1 (BAP1) alterations observed in 15% of U.S. tumors. Somatic, deleterious sequence changes occurred preferentially in U.S. Caucasian as compared to Chinese BCa patient tumors; are associated with papillary histologic features in a subset of tumors; and contribute to a significant number of tumors (10/14, 71%) that displayed alteration of genes encoding BRCA DNA repair pathway proteins. Genes encoding BRCA pathway proteins, including BAP1, BRCA1, BRCA2, PALB2, and ATM were altered by a combination of somatic and rare germline variants. We identified altered genes associated with clinical characteristics of BCa, including ‘immortalization' (TERT), aneuploidy (STAG2), proliferation (KDM6A), and papillary features in tumors (BAP1). STAG2 alterations are associated with a reduced patient survival, indicating a lethal subtype of disease; and altered X chromosome genes (STAG2 and KDM6A) are single copy in males and likely contribute to the cancer gender bias. Finally, we identified BRCA pathway alterations in BCa that are similar to those in breast, ovarian, prostate and kidney tumors, that have a common DNA repair deficiency that can be exploited by therapy targeting poly (ADP ribose) polymerase. Citation Format: Mike Nickerson, Kate M. Im, Guangwu Guo, Yaoting Gui, Sevilay Turan, James C. Costello, Quan Zhou, Zhiming Cai, Song Wu, M. Scott Lucia, Lee E. Moore, Michael Dean, Dan Theodorescu. Gene alterations associated with clinical characteristics of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A52.

Indoxyl Sulfate Enhance the Hypermethylation of Klotho and Promote the Process of Vascular Calcification in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a state of Klotho deficiency. The Klotho expression may be suppressed due to DNA hypermethylation in cancer cells so we have investigated the effects and possible mechanisms by which Klotho expression is regulated in human aortic smooth muscle cells (HASMCs). The vascular Klotho hypermethylation in radial arteries of patients with end-stage renal disease was described. Cultured HASMCs and 5/6-nephrectomized Sprague Dawley (SD) rats treated with indoxyl sulfate (IS) were used as in vitro and in vivo models, respectively. IS increased CpG hypermethylation of the Klotho gene and decreased Klotho expression in HASMCs, and potentiated HASMCs calcification. The expression of DNA methyltransferase (DNMT) 1 and 3a in HASMCs treated with IS was significantly increased and specific inhibition of DNA methyltransferase 1 by 5-aza-2'-deoxycytidine(5Aza-2dc) caused demethylation of the Klotho gene and increased Klotho expression. In rats, injection of IS potentiated vascular calcification, increased CpG hypermethylation of the Klotho gene and decreased Klotho expression in the aortic medial layer and all of these changes could be reverted by 5Aza-2dc treatment. Transcriptional suppression of vascular Klotho gene expression by IS and epigenetic modification of Klotho by IS may be an important pathological mechanism of vascular calcification in CKD.

The transcribed-ultraconserved regions in prostate and gastric cancer: DNA hypermethylation and microRNA-associated regulation.

The transcribed-ultraconserved regions (T-UCRs) are a novel class of non-coding RNAs, which are absolutely conserved (100%) between the orthologous regions of the human, rat and mouse genomes. Previous studies have described that several T-UCRs show differential expressions in cancers and might be involved in cancer development. We investigated the transcriptional levels of representative 26 T-UCRs and determined the regions that were differently expressed in prostate cancer (PCa) and gastric cancer (GC). A quantitative reverse transcription-polymerase chain reaction analysis revealed the downregulation of Uc.158+A expression by a DNA methylation-associated mechanism, which was restored by 5-Aza-dC (5-aza-2'-deoxycytidine) treatment. Bisulfite genomic sequencing using cell lines and tissue samples demonstrated cancer-specific CpG hypermethylation in both GC and PCa. However, Uc.416+A was only overexpressed in GC and we identified an miR-153 binding site in the possible regulatory region of Uc.416+A using online databases. Along with a forced expression or knockdown of miR-153 in MKN-74 GC cells, the transcriptional levels of Uc.416+A were significantly disturbed. A luciferase reporter gene assay supported the direct regulation of Uc.416+A expression by miR-153. Furthermore, Uc.416+A was associated with cell growth through the regulation of IGFBP6 (insulin-like growth factor-binding protein 6) in GC. These findings suggest an oncogenic role of Uc.416+A in GC, which suggests that our approach would provide new insights into functional studies of T-UCRs in cancer biology.

Constitutive expression of AhR and BRCA-1 promoter CpG hypermethylation as biomarkers of ERα-negative breast tumorigenesis.

BackgroundOnly 5–10 % of breast cancer cases is linked to germline mutations in the BRCA-1 gene and occurs early in life. Conversely, sporadic breast tumors, which represent 90-95 % of breast malignancies, have lower BRCA-1 expression, but not mutated BRCA-1 gene, and tend to occur later in life in combination with other genetic alterations and/or environmental exposures. The latter may include environmental and dietary factors that activate the aromatic hydrocarbon receptor (AhR). Therefore, understanding if changes in expression and/or activation of the AhR are associated with somatic inactivation of the BRCA-1 gene may provide clues for breast cancer therapy.MethodsWe evaluated Brca-1 CpG promoter methylation and expression in mammary tumors induced in Sprague–Dawley rats with the AhR agonist and mammary carcinogen 7,12-dimethyl-benzo(a)anthracene (DMBA). Also, we tested in human estrogen receptor (ER)α-negative sporadic UACC-3199 and ERα-positive MCF-7 breast cancer cells carrying respectively, hyper- and hypomethylated BRCA-1 gene, if the treatment with the AhR antagonist α-naphthoflavone (αNF) modulated BRCA-1 and ERα expression. Finally, we examined the association between expression of AhR and BRCA-1 promoter CpG methylation in human triple-negative (TNBC), luminal-A (LUM-A), LUM-B, and epidermal growth factor receptor-2 (HER-2)-positive breast tumor samples.ResultsMammary tumors induced with DMBA had reduced BRCA-1 and ERα expression; higher Brca-1 promoter CpG methylation; increased expression of Ahr and its downstream target Cyp1b1; and higher proliferation markers Ccnd1 (cyclin D1) and Cdk4. In human UACC-3199 cells, low BRCA-1 was paralleled by constitutive high AhR expression; the treatment with αNF rescued BRCA-1 and ERα, while enhancing preferential expression of CYP1A1 compared to CYP1B1. Conversely, in MCF-7 cells, αNF antagonized estradiol-dependent activation of BRCA-1 without effects on expression of ERα. TNBC exhibited increased basal AhR and BRCA-1 promoter CpG methylation compared to LUM-A, LUM-B, and HER-2-positive breast tumors.ConclusionsConstitutive AhR expression coupled to BRCA-1 promoter CpG hypermethylation may be predictive markers of ERα-negative breast tumor development. Regimens based on selected AhR modulators (SAhRMs) may be useful for therapy against ERα-negative tumors, and possibly, TNBC with increased AhR and hypermethylated BRCA-1 gene.

Epigenetic Repression of miR-218 Promotes Esophageal Carcinogenesis by Targeting ROBO1

miR-218, consisting of miR-218-1 at 4p15.31 and miR-218-2 at 5q35.1, was significantly decreased in esophageal squamous cell carcinoma (ESCC) in our previous study. The aim of this study was to determine whether aberrant methylation is associated with miR-218 repression. Bisulfite sequencing analysis (BSP), methylation specific PCR (MSP), and 5-aza-2′-deoxycytidine treatment assay were applied to determine the methyaltion status of miR-218 in cells and clinical samples. In vitro assays were performed to explore the role of miR-218. Results showed that miR-218-1 was significantly CpG hypermethylated in tumor tissues (81%, 34/42) compared with paired non-tumor tissues (33%, 14/42) (p < 0.05). However, no statistical difference was found in miR-218-2. Accordingly, expression of miR-218 was negatively correlated with miR-218-1 methylation status (p < 0.05). After demethylation treatment by 5-aza-2′-deoxycytidine, there was a 2.53- and 2.40-fold increase of miR-218 expression in EC109 and EC9706, respectively. miR-218 suppressed cell proliferation and arrested cells at G1 phase by targeting 3′ untranslated region (3′UTR) of roundabout guidance receptor 1 (ROBO1). A negative correlation was found between miR-218 and ROBO1 mRNA expression in clinical samples. In conclusion, our results support that aberrant CpG hypermethylation at least partly accounts for miR-218 silencing in ESCC, which impairs its tumor-suppressive function.

Epigenetic inactivation of galanin and GALR1/2 is associated with early recurrence in head and neck cancer.

The aim of this study was to investigate the prognostic value of galanin (GAL) and galanin receptor (GALR) promoter hypermethylation in patients with head and neck squamous cell carcinoma (HNSCC). The methylation status of three genes-GAL, GALR1, and GALR2 was examined in HNSCC patient tumors using quantitative methylation-specific PCR (Q-MSP). To determine the prognostic value of GAL, GALR1 and GALR2 methylation status, their associations with various clinical characteristics and patient survival were assessed in HNSCC patient tumors (n=142). Aberrant methylation of at least one gene was observed in 84 of the 142 (59.2%) primary tumors analyzed. The methylation index, defined as the ratio between the number of methylated genes and the number of genes examined, was positively correlated with larger tumor size (P=0.034) and disease recurrence (P<0.001). In the multivariate logistic-regression analysis, methylation of both GAL and GALR1 exhibited the highest association with poor survival (hazard ratio, 6.83, P=0.002). Moreover, among patients without lymph node metastasis, a multivariate analysis showed a significant trend for poor survival as the number of hypermethylated genes increased (log-rank test, P=0.003). CpG hypermethylation is a likely mechanism of GAL and GALR1/2 gene inactivation, indicating that GAL and its receptors play a role in HNSCC tumorigenesis. As such, GAL and GALR1/2 methylation status may serve as an important biomarker for clinical outcome.

Abstract A1-01: Translational genomics of urologic cancer genes

Abstract Next generation sequencing (NGS) of urologic tumor genomes has led to the detection of frequently altered cancer genes. We have characterized bladder (BCa), prostate (PCa), and kidney (RCC) tumor genomes using exome, whole genome, and transcriptome NGS; copy number and SNP arrays, and bisulfite sequencing for methylation to detect genomic alterations associated with disease. We recently examined BCa tumors from 54 U.S. patients and compared these to 99 Chinese patients to identify novel alterations of cancer genes encoding proteins associated with DNA and chromatin, including telomerase (TERT), stromal antigen 2 (STAG2), and BRCA1-associated protein-1 (BAP1). We are the first to show the TERT promoter is altered in BCa by both somatic and germline nucleotide changes in 37/54 (69%) and 30/54 (56%) tumors, respectively; and that TERT somatic alterations do not correlate with other cancer gene mutations examined by exome NGS. Most TERT germline variants were novel (19/20), and three variants were confirmed as both somatic and germline in distinct tumors, including the most frequent variant, c. – 245 T&amp;gt;C (rs2853669). We show somatic promoter alterations are associated with increased TERT expression but, paradoxically, with significantly shorter telomeres in tumors as compared to matched normal tissue. STAG2 was altered by somatic deleterious sequence changes, genomic deletions, and promoter CpG hypermethylation. Mutations are associated with reduced patient survival and tumor cell aneuploidy based on an increased number of chromosomal arm copy number changes. Somatic BAP1 mutations are primarily deleterious sequence changes that occurred preferentially in U.S. Caucasian as compared to Chinese BCa patients. BAP1 mutations are associated with papillary histologic features in a subset of tumors and with mutations in the histone lysine–specific demethylase 6A (KDM6A/UTX) gene. BAP1 mutations contribute to a significant number of tumors with somatic and rare germline variants in BRCA pathway genes, including BRCA1, BRCA2, PALB2, and ATM. We experimentally examined the function of KDM6A since it is the most frequently altered chromatin modifying gene in 24% of tumors. KDM6A loss in human BCa cells enhanced in vitro proliferation, in vivo tumor growth, and cell migration, confirming KDM6A loss drives the BCa phenotype. A comparison of BCa and other urologic tumor genomes allows us to identify several emergent common characteristics. Significant numbers of tumors are altered by rare, disease-associated germline cancer gene variants that likely have substantive effects on cancer risk and which likely influence the subsequent accrual of somatic alterations. These likely contribute to the missing heritability that is observed by cancer epidemiology studies. Many cancer genes are altered by characteristic frequencies of nonsynonymous sequence changes, promoter variants, copy number variation, and hypermethylation, that are routinely observed for a given gene-tissue-patient ethnicity combination. Approximately 4-5 tumor suppressors are altered for each oncogene, and interactions between altered cancer genes affect the resulting biological functions. In BCa, we show KDM6A loss increases cell proliferation which likely results in shorter telomeres even though TERT expression is increased. We identified altered cancer genes such as STAG2 that are associated with discrete clinical aspects of the disease phenotype, namely aneuploidy and reduced patient survival indicating a lethal subtype of disease. Cancer genes on the X chromosome such as STAG2 and KDM6A have a single copy in men and likely contribute to the gender bias observed in cancer. Finally, cancer gene alterations can identify patients likely to respond to a currently approved therapy. BAP1 and BRCA pathway alterations define BCa, PCa, and RCC tumors with a common DNA repair deficiency that might be exploited by targeted therapies such as poly (ADP ribose) polymerase inhibitors. Citation Format: Michael L. Nickerson, Kate M. Im, Sevilay Turan, Guangwu Guo, Lee E. Moore, Dan Theodorescu, Michael Dean. Translational genomics of urologic cancer genes. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-01.