Abstract Hepatocellular carcinoma (HCC), the most prevalent type of primary liver cancer, is the second leading cause of cancer death worldwide. It is estimated that early HCC detection would increase the cure rate from 5% to 80%. Approximately 85% of individuals with HCC have underlying liver cirrhosis which is the main risk factor for developing HCC. Every year up to 5% of cirrhotic patients are diagnosed with HCC. The biggest challenge that physicians face today is to distinguish cirrhotic patients that will develop cancer from those that will not. Thus, early detection biomarkers that can be implemented as screening tools in populations at risk are of high interest. In our previous genome-wide study, we demonstrated that blood DNA has different DNA methylation patterns specific to HCC samples collected before diagnosis (pre-diagnostic) compared to healthy individuals without cancer. We established 10 differentially methylated probes corresponding to 10 genes that distinguish pre-diagnostic HCC blood samples from healthy controls. In the current study using pyrosequencing, we tested the diagnostic potential of these probes in blood samples from a prospective cohort of cirrhotic patients without HCC at the time of blood collection. Twenty eight of these patients developed HCC within 4 years of follow-up. These 28 cases were matched with 23 cirrhotic controls on gender, age, ethnicity, hepatitis C, and diabetes. Five out of the 10 tested probes distinguished cirrhotic patients who subsequently developed HCC (cases) from those who stayed cancer free (controls) (p<0.05, Mann-Whitney U test). The highest differences were observed at CpG sites located within BARD1 whose methylation was consistently lower in cases vs. controls. Using this probe, 22 cases were correctly identified as cases (79% sensitivity), whereas only 6 controls were falsely included into cases (74% specificity). Analysis of the panel of the 5 probes increased sensitivity to 86% with a threshold of 2 positive probes, while maintaining 74% specificity. Receiver Operating Characteristics (ROC) curve analysis confirmed high predictive value for the 5 probes in the cirrhotic population [Area Under the Curve (AUC) = 0.80]. The established DNA methylation biomarkers have better values of performance compared to current recommended methods for HCC surveillance, such as serum alpha-fetoprotein (AFP) that can miss up to 40% of patients. Our results establish the possible diagnostic value of gene-specific DNA methylation in blood DNA for HCC early detection in populations at risk such as individuals with liver cirrhosis. This study was supported by Showalter Trust and PCCR Awards granted to BS. Citation Format: Katarzyna Lubecka, Lucinda Kurzava, Kirsty Flower, Hannah Buvala, Samer Gawrieh, Suthat Liangpunsakul, Naga Chalasani, James M. Flanagan, Barbara Stefanska. Differences in gene-specific DNA methylation in blood DNA as a biomarker for early detection of hepatocellular carcinoma in populations at risk. [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 2786.
Late onset of clinical symptoms in hepatocellular carcinoma (HCC) results in late diagnosis and poor disease outcome. Approximately 85% of individuals with HCC have underlying liver cirrhosis. However, not all cirrhotic patients develop cancer. Reliable tools that would distinguish cirrhotic patients who will develop cancer from those who will not are urgently needed. We used the Illumina HumanMethylation450 BeadChip microarray to test whether white blood cell DNA, an easily accessible source of DNA, exhibits site-specific changes in DNA methylation in blood of diagnosed HCC patients (post-diagnostic, 24 cases, 24 controls) and in prospectively collected blood specimens of HCC patients who were cancer-free at blood collection (pre-diagnostic, 21 cases, 21 controls). Out of 22 differentially methylated loci selected for validation by pyrosequencing, 19 loci with neighbouring CpG sites (probes) were confirmed in the pre-diagnostic study group and subjected to verification in a prospective cirrhotic cohort (13 cases, 23 controls). We established for the first time 9 probes that could distinguish HBV-negative cirrhotic patients who subsequently developed HCC from those who stayed cancer-free. These probes were identified within regulatory regions of BARD1, MAGEB3, BRUNOL5, FXYD6, TET1, TSPAN5, DPPA5, KIAA1210, and LSP1. Methylation levels within DPPA5, KIAA1210, and LSP1 were higher in prospective samples from HCC cases vs. cirrhotic controls. The remaining probes were hypomethylated in cases compared with controls. Using blood as a minimally invasive material and pyrosequencing as a straightforward quantitative method, the established probes have potential to be developed into a routine clinical test after validation in larger cohorts.
DNA hypomethylation was previously implicated in cancer progression and metastasis. The purpose of this study was to examine whether stilbenoids, resveratrol and pterostilbene thought to exert anticancer effects, target genes with oncogenic function for de novo methylation and silencing, leading to inactivation of related signaling pathways. Following Illumina 450K, genome-wide DNA methylation analysis reveals that stilbenoids alter DNA methylation patterns in breast cancer cells. On average, 75% of differentially methylated genes have increased methylation, and these genes are enriched for oncogenic functions, including NOTCH signaling pathway. MAML2, a coactivator of NOTCH targets, is methylated at the enhancer region and transcriptionally silenced in response to stilbenoids, possibly explaining the downregulation of NOTCH target genes. The increased DNA methylation at MAML2 enhancer coincides with increased occupancy of repressive histone marks and decrease in activating marks. This condensed chromatin structure is associated with binding of DNMT3B and decreased occupancy of OCT1 transcription factor at MAML2 enhancer, suggesting a role of DNMT3B in increasing methylation of MAML2 after stilbenoid treatment. Our results deliver a novel insight into epigenetic regulation of oncogenic signals in cancer and provide support for epigenetic-targeting strategies as an effective anticancer approach.
Abstract Alterations in DNA methylation occur at different stages of cancer, including initiation, and may underlie up-regulation of genes with oncogenic functions. The NOTCH pathway is often overactive in breast cancer and plays roles in cancer development and progression. It is therefore a possible target for anti-cancer strategies. However, the mechanisms of NOTCH regulation in mammary carcinogenesis remain unclear which hinders the development of effective approaches to target this oncogenic pathway. Interestingly, certain dietary compounds such as polyphenols with a stilbenoid structure suppress the NOTCH signals in cancer and were shown in our study to modify epigenetic marks in genes positively regulating the pathway, including MAML2. In the present study, using two polyphenols, resveratrol from grapes and pterostilbene from blueberries, we investigate the implication of DNA methylation in regulation of NOTCH in breast cancer. Non-invasive MCF10CA1h and invasive MCF10CA1a human breast cancer cell lines were used as an experimental model. Following genome-wide DNA methylation analysis with Illumina 450K BeadChip array, pyrosequencing and QPCR were performed to assess methylation and expression of MAML2 and NOTCH target genes. Chromatin immunoprecipitation (ChIP) was applied to determine binding of epigenetic enzymes to MAML2 regulatory region. Depletion with siRNA was applied to establish functional link between MAML2 silencing and NOTCH activity. We found 4,293 CpG sites differentially methylated upon 9-day treatment of MCF10CA1h breast cancer cells with 15µM resveratrol as compared to untreated cells (0.05<differential methylation<-0.05, p<0.03, Wilcoxon rank-sum test). Among these changes, 3,508 CpGs corresponding to 1,707 genes were hypermethylated and functionally associated with oncogenic signaling pathways, including NOTCH. As the array data indicated, resveratrol led to re-methylation of enhancer region of MAML2 that is a coactivator of NOTCH target genes. The 15-20% increase in methylation of MAML2 was confirmed by pyrosequencing in breast cancer cells with both high and low invasive potential. Pterostilbene, an analog of resveratrol, exerted similar effects on MAML2 methylation state at 10µM concentration. Along with methylation of the MAML2 enhancer region, the compounds decreased MAML2 expression by 30-60%. Stronger MAML2 down-regulation was achieved in invasive cancer cells as compared to non-invasive cells after exposure to either resveratrol or pterostilbene. Epigenetic silencing of MAML2 was associated with downregulation of NOTCH target genes, including HES1 (30-50%), HEY1 (30-60%), and NOTCH1 (40-65%). Depletion of MAML2 with siRNA mimicked polyphenols' effects leading to suppression of NOTCH pathway. It supports a functional link between MAML2 and NOTCH signaling. The most profound effects were observed in invasive MCF10CA1a cells where further analyses revealed 2-fold increase in trimethylation of H3K27 (repressive histone mark) and 30% decrease in acetylation of H3K9 (activating mark) at MAML2 enhancer region upon exposure to resveratrol. The condensed chromatin structure was associated with binding of DNMT3B to the tested fragment of MAML2 upon polyphenol treatment whereas no binding was detected in untreated cells. It suggests the role of DNMT3B in increased methylation of MAML2, which is consistent with increased DNMT3B expression upon treatment with polyphenols. Our results establish a role for epigenetic modifications in regulation of NOTCH oncogenic signaling in breast cancer. It constitutes a novel insight into regulation of oncogenic signals in cancer and provides support for epigenetic-targeting strategies as an effective anti-cancer approach. This study was supported by the Purdue University Center for Cancer Research, Indiana CTSI (UL1TR001108), and Women's Global Health Institute granted to BS. Citation Format: Katarzyna Lubecka, Lucinda Kurzava, Kirsty Flower, Hannah Buvala, Dorothy Teegarden, Ignacio Camarillo, James M. Flanagan, Barbara Stefanska. Epigenetic regulation of NOTCH oncogenic signaling in breast 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 B17.
Abstract Hepatocellular carcinoma (HCC), one of the most prevalent types of primary liver cancer, is the sixth most common cancer worldwide and the third leading cause of cancer death with rising mortality and morbidity rates. As late onset of HCC accounts for late diagnosis and poor prognosis and early detection increases cure rate from 5% to 80%, identifying reliable and quantifiable biomarkers of risk prediction is of high interest. Aberrations in the DNA methylation patterns, an important early event in carcinogenesis, have been shown to differentiate HCC tumors from normal tissues. However, these changes as predictive markers would have a high application in clinics only if detectable by minimally invasive tests like blood test. In the present study, we performed a comprehensive evaluation of DNA methylation profiles in blood DNA collected before diagnosis with HCC. Aberrant methylation was investigated in DNA isolated from blood of 21 HCC patients (cases) who provided samples between 1-4 years prior to diagnosis and 21 controls enrolled by the Indiana Biobank of Indiana CTSI. Cases were matched with controls for gender, age, ethnicity, hepatitis C infection, and diabetes. We used Infinium Human Methylation 450K BeadChip array for genome-wide DNA methylation analysis and pyrosequencing for validation of DNA methylation differences. We found 966 probes differentially methylated between cases and controls with p<0.05 and intraclass correlation coefficient >0.5. Among these significant changes, 732 CpG sites are hypomethylated in HCC cases compared to matched controls and include 130 CpGs corresponding to 75 genes with delta beta value (differential methylation) ≥0.1. Forty six CpG sites out of 234 significantly hypermethylated probes show delta beta value ≥0.1 and correspond to 46 genes. Functional analyses using GO, KEGG and DAVID knowledgebase indicates that hypomethylated genes are associated with Wnt signaling, cell adhesion, blood coagulation, and regulation of transcription, whereas hypermethylated genes are enriched with cytoskeleton organization and small GTPase mediated signal transduction. One of the genes hypomethylated in blood DNA of HCC cases prior to diagnosis is TET1 that was found to be 8-fold overexpressed in HCC tumors in our previous studies and implicated in gene-specific hypomethylation. Interestingly, 75% of differentially methylated sites in blood DNA of HCC cases are hypomethylated prior to diagnosis. Validation by pyrosequencing of four hypomethylated probes including CpGs in the CpG island shore of BRUNOL5 that was linked to fatty liver disease in earlier reports and found demethylated and overexpressed in HCC tumors, suggests their predictive potential. Our results establish the possible predictive value of aberrant methylation, in particular DNA hypomethylation, in blood DNA for risk of HCC. This study was supported by Showalter Trust and Purdue Center for Cancer Research Awards granted to BS. Citation Format: Katarzyna Lubecka-Pietruszewska, Lucinda Kurzava, Hannah Buvala, Kirsty Flower, Samer Gawrieh, Jennifer Mansfield, Naga Chalasani, James M. Flanagan, Barbara Stefanska. Differential DNA methylation in peripheral blood DNA as a biomarker of hepatocellular carcinoma risk. [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 2959. doi:10.1158/1538-7445.AM2015-2959
Late onset of clinical symptoms in hepatocellular carcinoma (HCC) results in late diagnosis and poor disease outcome. Approximately 85% of individuals with HCC have underlying liver cirrhosis. However, not all cirrhotic patients develop cancer. Reliable tools that would distinguish cirrhotic patients who will develop cancer from those who will not are urgently needed. We used the Illumina HumanMethylation450 BeadChip microarray to test whether white blood cell DNA, an easily accessible source of DNA, exhibits site-specific changes in DNA methylation in blood of diagnosed HCC patients (post-diagnostic, 24 cases, 24 controls) and in prospectively collected blood specimens of HCC patients who were cancer-free at blood collection (pre-diagnostic, 21 cases, 21 controls). Out of 22 differentially methylated loci selected for validation by pyrosequencing, 19 loci with neighbouring CpG sites (probes) were confirmed in the pre-diagnostic study group and subjected to verification in a prospective cirrhotic cohort (13 cases, 23 controls). We established for the first time 9 probes that could distinguish HBV-negative cirrhotic patients who subsequently developed HCC from those who stayed cancer-free. These probes were identified within regulatory regions of BARD1, MAGEB3, BRUNOL5, FXYD6, TET1, TSPAN5, DPPA5, KIAA1210, and LSP1. Methylation levels within DPPA5, KIAA1210, and LSP1 were higher in prospective samples from HCC cases vs. cirrhotic controls. The remaining probes were hypomethylated in cases compared with controls. Using blood as a minimally invasive material and pyrosequencing as a straightforward quantitative method, the established probes have potential to be developed into a routine clinical test after validation in larger cohorts.
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