Extent Of Promoter Methylation Research Articles

Abstract A02: The HER4 BH3-only protein and tumor suppressor is epigenetically silenced in endocrine refractory and triple negative breast cancers

Abstract Breast cancer is the most commonly diagnosed cancer in North American women and remains the second most common cause of cancer related deaths in women. In contrast to the mitogenic activities of the EGFR-family members EGFR itself, HER2, and HER3; HER4, the fourth and final member of the EGFR-family to be discovered, suppresses breast tumor cell growth. HER4 is also unique because it is the only member of the EGFR family to undergo proteolytic cleavage at the cell surface to generate an independently signaling intracellular domain (4ICD). Clinically, HER4 expression is extinguished during breast tumor progression and patients with tumors lacking HER4 expression have significantly worse prognosis. We investigated the apparent tumor suppressor activity of HER4 and mechanisms of HER4 suppression during breast tumor progression. We found that 4ICD harbors a pro-apoptotic BH3-only domain required for its tumor cell killing activity. To evade the potent tumor cell killing activity of 4ICD, tumor cells acquire mechanisms to suppress HER4 expression. Importantly, we identified a CpG island within the HER4 promoter and show by pyrosequencing of bisulfite treated DNA an inverse correlation between HER4 expression and the extent of promoter methylation. Clinically, increased levels of HER4 promoter methylation were significantly associated with the most aggressive breast cancer subtypes (hormone refractory, HER2 positive, and triple negative) and significantly worse patient prognosis. Consistent with these clinical findings, the HER4 promoter is hypermethylated and HER4 expression is suppressed in multiple endocrine resistant and triple negative breast cancer cell lines. Reestablishing, by ectopic expression, or reactivating, through 5-aza-2-deoxycytidine treatment, HER4 expression results in dramatic BH3-dependent apoptosis of both endocrine resistant and triple negative breast cancer cell lines. To identify alterations in epigenetic regulators common to multiple breast tumor cell lines with suppressed HER4 expression we used Qiagen RT-Profiler PCR Arrays to analyze expression of 168 key chromatin modifying enzymes and remodeling factors. The most dramatic alterations common to two endocrine resistant and three triple negative breast tumor cell lines were suppression of the chromatin remodeling protein CHD5 and suppression of the TET2 deoxygenase - which initiates a DNA demethylation pathway. We are currently investigating the impact of modulated CHD5 and TET2 expression on the reactivation of HER4 cell killing activity in endocrine resistant and triple negative breast tumors. In conclusion, our data supports a pro-apoptotic tumor suppressor function for HER4 which is epigenetically suppressed in endocrine refractory and triple negative breast tumors through promoter hypermethylation. Reactivation of epigenetically silenced HER4 results in dramatic tumor cell apoptosis, providing a therapeutic strategy for the treatment of the most aggressive forms of breast cancer. Note: This abstract was not presented at the conference. Citation Format: Wen Han, Eleanor Semmes, Frank E. Jones. The HER4 BH3-only protein and tumor suppressor is epigenetically silenced in endocrine refractory and triple negative breast cancers. [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 A02.

Expression of O<sup>6</sup>-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions, depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included (a) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, (b) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and (c) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumor-selective reduction in MGMT activity exist in human tissue.

Alterations in methylation status of immune response genes promoters in cell-free DNA during a hemodialysis procedure

Objective: Elevations of cell-free DNA (cfDNA) concentrations during hemodialysis (HD) sessions were reported in numerous studies regardless of an applied therapeutic protocol. It is generally thought that the elevated concentrations represent the consequence of apoptosis on the dialysis membranes. No data concerning the qualitative characteristics of cfDNAs in HD patients have been published till today; therefore, we focus on the promoter methylation status of genes involved in immune response.Research design and methods: We isolated cfDNA from randomly selected patients before and after a HD session and from healthy subjects examined two times per day with 4 h interval. The extent of promoter methylation of 24 genes involved in immune response was examined using the ‘EpiTect Methyl qPCR Array Inflammatory Response and Autoimmunity’.Results: In our pilot study, we discovered significant changes in methylation patterns of genes IL-7, IL-13, IL-17C and TYK2 between HD patients and healthy subjects.Conclusion: Methylation of immune response genes promoters may be detected using EpiTect Methyl qPCR Array Inflammatory Response and Autoimmunity at the level of cfDNA to provide the information about the actual state of immune response in HD patients.

Abstract 2221: DNA methylation of microRNA genes in gastric carcinoma and its clinicopathological association

Abstract Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric carcinoma (GC). DNA methylation of multiple CpG sites in promoter region of several tumor-related genes, such as hMLH1, p14, p16, and APC has been analyzed in GC so far. MicroRNAs (miRNA) are small, non-coding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies indicate that epigenetic silencing of miRNAs with tumor suppressor activity by CpG island methylation plays an important role in gastric carcinogenesis. To explore the role of epigenetic mechanisms in the down-regulation of miRNA genes (has-miR-9, has-miR-129, has-miR-137), we examined the presence of DNA methylation-associated silencing of miRNAs in GC and observed that aberrant methylation of these miRNAs is associated with expression of target gene products. Materials and Methods: The extent of promoter methylation of 4 miRNA genes (has-miR-9-1, has-miR-9-3, has-miR-129-2, has-miR-137) was assessed using methylation-specific polymerase chain reaction (MSP) in 100 GC tissues (50 early gastric cancers and 50 advanced gastric cancers) and corresponding non-tumor tissues. Twenty patients with benign gastric pathology were also included as a control group. The potential target gene products of miRNAs were studied by immunohistochemistry and the relationship between methylation profile of miRNAs promoter and clinicopathological parameters was analyzed. Results: Methylation of the has-miR-9-3 and has-miR-137 CpG island was frequently observed in tumor tissues (89% and 86%, respectively) and non-tumor tissues in 100 GC patients (70% and 78%), but not in normal gastric tissues (55% and 55%). Methylation of the has-miR-9-1 is slightly higher in tumor tissues (65%) than normal gastric tissue (45%). However, methylation level of the has-miR-129-2 was not shown significant difference in tumor tissues (97%) compared with non-tumor tissues (90%) and normal gastric tissues (90%). Expression of NF-κB and SOX4 protein, which are has-miR-9 and has-miR-129-2 potential target respectively, were inversely correlated with methylation level of miRNAs. The presence of H. pylori infection was significantly associated with aberrant methylation of miRNA genes (p<0.05). But, there was no significant correlation between aberrant methylation of miRNAs and other clinicopathological factors. Conclusion: These results suggest that specific miRNAs methylation in GC could be an important molecular mechanism causing loss of control on its target, and it may be correlated with the high transcriptional activity of target gene. Epigenetic silencing of some miRNAs may involve in the early stage of gastric carcinogenesis and could be used as an efficient diagnostic biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2221. doi:10.1158/1538-7445.AM2011-2221

Reactivation of epigenetically silenced HER4/ERBB4 results in apoptosis of breast tumor cells

Experimental and clinical data support a growth inhibitory role for HER4 in breast cancer. Clinically HER4 expression is extinguished during breast tumorigenesis supporting a tumor suppressor function for HER4, however, a molecular mechanism to explain the selective loss of HER4 expression has remained elusive. Epigenetic mechanisms, for example, aberrant gene promoter hypermethylation, have been shown to ablate tumor suppressor gene expression in breast carcinomas. We identified a CpG island within the HER4 promoter and show by pyrosequencing of bisulfite-treated DNA an inverse correlation between HER4 expression and the extent of promoter methylation. Treatment of the HER4-negative BT20 cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine (DAC)-enhanced HER4 expression, confirming a role for DNA methylation in suppressed HER4 expression. DAC treatment to reactive HER4 expression in combination with the HER4 ligand heregulin-β1 (HRG) resulted in apoptosis of BT20 cells providing a novel therapeutic strategy for triple-negative tumors. The BT20 cells were rescued from apoptosis when preincubated with HER4 small interfering RNA, thereby confirming a role for HER4 in DAC/HRG-induced apoptosis. We verified HER4 promoter methylation in primary breast carcinomas and detected a significant increase in HER4 promoter methylation in HER4-negative breast tumors (P<0.001). Furthermore, increased levels of HER4 promoter methylation were significantly associated with worse patient prognosis (P=0.0234). Taken together, our data support a tumor suppressor function for HER4, which is epigenetically suppressed in breast tumors through promoter hypermethylation.

Abstract 157: Quantitative COX2 promoter methylation analysis in gastric carcinoma

Abstract Background: Epigenetic silencing of tumor-related genes, due to CpG island methylation, is considered to be an important mechanism for the development of many tumors, including gastric cancinoma. DNA methylation of multiple CpG sites in promoter regions of several tumor related genes, such as hMLH1, p14, p16, and APC has reported in gastric carcinoma. Recent studies indicate that aberrant CpG island methylation, and subsequent silencing of the COX-2 promoter, has been observed in a subset of gastric carcinomas (GC). To determine the occurrence of CpG island hypermethylation in GC in relation to H. pylori infection, the status and level of CpG methylation in promoter region of COX-2 was analyzed in early and advanced gastric carcinomas as opposed to normal gastric tissues. Materials and Methods: The extent of promoter methylation of COX-2 gene was assessed quantitatively using Pyrosequencing (PS) in 60 early gastric cancers (EGC) and 60 advanced gastric cancers (AGC) samples harvested upon gastrectomy, and 40 normal gastric mucosa samples from patients with benign gastric pathology. Giemsa stain was performed to detect H. pylori in neoplastic and non-neoplastic gastric mucosa. Gene product was studied by immunohistochemistry and the relationship between methylation profile of gene promoter and clinicopathological parameters was analyzed. Results: The PS analysis of GCs revealed a high frequency of COX-2 methylation in 30.0% (36/120). The methylation frequency for COX-2 gene by PS techniques was significantly higher in EGCs than in AGCs (40.0% and 20.0%, respectively, p&amp;lt;0.05) There was a significant difference of COX-2 methylation in between GCs and normal gastric tissues (30.0% vs. 10.0%, respectively by PS, p&amp;lt;0.05). The mean methylation level in 8 CpG sites of COX-2 promoter by PS was 19.9%, 33.5%, and 18.5% in normal gastric tissue, EGCs, and AGCs respectively. COX-2 gene methylation was significantly associated with H. pylori infection (p&amp;lt;0.001). However, the methylation of COX-2 promoter by PS was not correlated with reduced or absent expression of COX-2 protein (p&amp;gt;0.05). Conclusion: we found that COX-2 promoter methylation was significantly higher in the tumor tissues and was early event for gastric carcinoma development, but not in progression of GC. These results suggest that COX-2 gene methylation may be important in the initial development of gastric carcinogenesis and the H. pylori infection is associated with promoter methylation of COX-2 gene. Furthermore, standard PCR followed by PS could be a more specific and quantitative method providing a more comprehensive picture of the distribution of DNA methylation throughout the promoter regions of specific genes, which will be of benefit in oncology research. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 157.

Abstract 3213: GRHL2 enhances hTERT transcription by inhibiting promoter DNA methylation at 5’ CpG island

Abstract We recently identified Grainyhead-like 2 (GRHL2), a mammalian homologue of Grainyhead in Drosophila, to be a novel transcription factor that binds to and regulates the activity of the human telomerase reverse transcriptase (hTERT) gene promoter. In the current study, we investigated the biological function of GRHL2 in normal human keratinocytes (NHK) and elucidated the molecular mechanism underlying GRHL2 regulation of hTERT gene expression. Primary NHK were infected with retroviral vectors expressing GRHL2 or the empty vector. GRHL2 transduction led to significant extension of the replicative lifespan of NHK by evading the senescence block. However, GRHL2 failed to cause immortalization of NHK, alone or in combination with Bmi-1, a polycomb group protein that bestows extended replication capacity to NHK. Enhanced proliferation of cells transduced with GRHL2 was also observed in three dimensional organotypic raft culture. Telomerase activity was detected in replicating NHK while it was rapidly lost during senescence. GRHL2 transduction increased the expression of hTERT and maintained detectable telomerase activity for extended time period, suggesting that GRHL2 positively influences the hTERT expression and telomerase activity in NHK. Upon knockdown of endogenous GRHL2, hTERT mRNA expression and telomerase activity were notably reduced. We previously showed that hTERT expression in NHK is regulated in part by promoter methylation at the 5’ CpG island. Thus, we determined the effects of GRHL2 on the methylation status of hTERT promoter by methylation-specific PCR (MSP). Loss of hTERT expression in the control NHK occurred with hTERT promoter hypermethylation. On the contrary, GRHL2 transduction notably reduced the extent of promoter methylation in NHK. This effect was not linked with enhanced cell proliferation because Bmi-1 transduction in NHK, which allowed prolonged cell proliferation, showed no effect on the hTERT promoter methylation. These data indicate that GRHL2 inhibits the promoter DNA methylation, thereby delaying hTERT gene inactivation and senescence of NHK. This study was supported by the grants R01DE18295, K02DE18959 from NIDCR/NIH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3213.

Abstract 4940: E-cadherin methylation is preceded in normal mucosa of diffuse type gastric carcinoma

Abstract Background: The importance of loss of E-cadherin (encoded by CDH1) to cancer progression is well established. CDH1 is mutationally inactivated in nearly half of diffuse type gastric cancer (DGC). Although CDH1 promoter methylation has been suggested to be a major second hit in hereditary diffuse gastric cancer, its role in sporadic DGCs is not clarified. Searching for the second hit methylation in potential precursor lesions in sporadic DGC, we investigated the CDH1 methylation frequency in a series of DGCs and matching normal mucosas and then compared the result with the CDH1 methylation frequency in normal mucosas from benign gastric pathology. Materials and Methods: The extent of promoter methylation of CDH1 gene was assessed quantitatively using Pyrosequencing in 72 DGCs with two matching normal mucosas from adjacent and remote foci, and 24 normal gastric mucosa samples from patients with benign gastric pathology. Gene product was studied by immunohistochemistry and the relationship between methylation profile of gene promoter and clinicopathological parameters was analyzed. Results: The Pyrosequencing analysis of DGCs revealed a high frequency of CDH1 methylation in 77.8% (56/72). The methylation frequency for CDH1 gene in matching normal mucosas from adjacent and remote foci was 70.8% (51/72) and 72.2% (52/72), respectively. According to the distance from the tumor location the result did not show significant difference. Moreover, total of 65 out of 72 cases (90.3%) showed at least one focus with CDH1 methylation. Frequency of CDH1 methylation in normal gastric mucosa samples with benign gastric pathology was 50.0% (12/24). There was a significant difference of CDH1 methylation in between normal mucosas from DGCs and normal mucosas with benign gastric pathology (90.3% vs. 50.0%, respectively, p&amp;lt;0.001). CDH1 methylation in matching normal gastric mucosas was counted as a statistically significant risk factor when adjusted for age and sex using a binary logistic regression model (p&amp;lt;0.001). Additionally loss or diminished expression of E-cadherin was confirmed by immunohistochemistry in 69 of 72 tumor samples (96.9%) and its down expression was not uncommonly observed in adjacent normal gastric mucosas. Conclusion: We found that CDH1 promoter methylation in the normal gastric mucosas from DGC samples was significantly higher than in the normal gastric mucosas with benign pathology but had a similar frequency with tumor tissues. These results suggest that CDH1 gene methylation may be important in the initial development of sporadic DGC tumorigenesis and the presence of DGC progenitor cells may be traced by the E-cadherin down-expression in normal gastric mucosa from different foci. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4940.

Epigenetically regulated clonal heritability of CTA expression profiles in human melanoma

The intratumoral heterogeneity of cancer testis antigens (CTA) expression, which is driven by promoter methylation status, may hamper the effectiveness of CTA-directed vaccination of melanoma patients. Thus, we investigated whether the intratumoral heterogeneity of CTA expression is inherited at cellular level, or evolves throughout cellular replication, leading to a phenotypically unstable tumor cell population with reduced immunogenicity and/or able to escape immune control. Utilizing a previously characterized ex vivo clonal model of intratumoral heterogeneity of CTA expression in melanoma, Mel 313 MAGE-A3-low clone 5 (clone 5(M3-low)) and MAGE-A3-high clone 14 (clone 14(M3-high)) were sub-cloned and analyzed for CTA profile. Molecular assays demonstrated that levels of MAGE-A3 expression were highly conserved among generated sub-clones, as compared to parental clones. A similar behavior was identified for an extensive panel of other CTA investigated. Inherited levels of MAGE-A3 expression correlated with the extent of promoter methylation among clone 5(M3-low) and clone 14(M3-high) sub-clones analyzed. Treatment of clone 5(M3-low) with a DNA hypomethylating agent (DHA) resulted in an up-regulated expression of MAGE-A3, which was inherited at single cell level, being still detectable at day 60 in its sub-clones. Bisulfite sequencing demonstrated that also MAGE-A3 promoter methylation status was inherited among sub-clones generated from DHA-treated clone 5(M3-low) and strictly correlated with MAGE-A3 expression levels in investigated sub-clones. Similar results were obtained for additional CTA studied. Altogether our findings demonstrate that constitutive and DHA-modified CTA profiles of melanoma cells are clonally inherited throughout cellular replications, thus providing relevant insights to improve the effectiveness of CTA-based immunotherapy.

Lack of death receptor 4 (DR4) expression through gene promoter methylation in gastric carcinoma

To determine the underlying mechanism for the differential expression, the extent of promoter methylation in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-related genes acting downstream of TRAIL was examined in early and advanced gastric carcinomas. The extent of promoter methylation in the DR4, DR5, DcR1, DcR2, and CASP8 genes was quantified using bisulfite modification and methylation-specific polymerase chain reaction. The promoters for DcR1, DcR2, and CASP8 were largely unmethylated in early gastric carcinoma, advanced gastric carcinoma, and controls, with no significant difference among them. Protein levels of DR4, DcR1, and DcR2 as revealed by immunohistochemistry correlated with the extent of the respective promoter methylation (P < 0.05 in all cases). Hypomethylation, rather than hypermethylation, of the DR4 promoter was noted in invasive gastric malignancies, with statistical significance (P = 0.003). The promoter methylation status of TRAIL receptors in gastric carcinoma may have clinical implications for improving therapeutic strategies in patients with gastric carcinoma.

Comparative Promoter Methylation Analysis of p53 Target Genes in Urogenital Cancers

Introduction: The methylation status of selected new p53 target genes in bladder, kidney and testicular cancer was investigated to find similarities in methylation frequency and individual levels of methylation with regard to the histopathological classification and biological behavior. Material and Methods: The patients’ samples were evaluated using real-time methylation-specific PCR (MSP) investigating the methylation status of APAF-1, CASP-8, DAPK-1 and IGFBP-3. Included were 103 patients with bladder cancer, 85 patients with kidney cancer and 42 patients with testicular cancer in non-advanced and advanced tumor stages. Non-cancerous tissue from 49 patients was used as control. Results: Methylation frequency was highest for APAF-1 (88–100%) and DAPK-1 (17–86%) through all the different tumor types. Highest APAF-1 and DAPK-1 levels of methylation were found in the advanced tumor groups. In normal tissue, methylation frequency and methylation levels were significantly lower. APAF-1 methylation was also frequent in normal testicular tissue but to lower methylation levels as in cancerous tissue. APAF-1 and IGFBP-3 methylation levels were significantly higher in recurrent superficial and muscle-invasive bladder tumors. Methylation levels of APAF-1 also correlated with higher risk for recurrent metastatic disease in non-advanced tumors of the kidney. Conclusions: The methylation profile observed demonstrates a substantial role of the APAF-1 gene in tumorigenesis. The different methylation pattern in testicular cancer might represent a different methylotype of this cancer. The extent of promoter methylation may be a promising target for application as a tumor marker in urogenital cancer disease.