Detection of Serum Free DNA Hypermethylation in Surgically Resected Adenocarcinoma of the Lung

Park, Sun Jung,Kim, Young Tae,Park, Ju-Yeon,Wi, Hyun Cho,Kang, Chang Hyun,Sung, Sook-Whan,Kim, Joo Hyun 대한폐암연구회 2008 Journal of Lung Cancer Vol.7 No.2

Purpose: Aberrant DNA methylation patterns have been commonly associated with human cancers. We have investigated the frequency of DNA hypermethylation in promoter regions from adenocarcinomas of the lung and then attempted to detect the same epigenetic changes from patient serum samples. Materials and Methods: We collected tissues from 72 cases of lung adenocarcinomas. The cancer and normal lung tissues were tested for DNA hypermethylation using methylation-specific PCR (MSP). The genes investigated were DAPK, RARβP2 and p16. We selected 12 patients where promoter hypermethylation was present for all three genes and four patients where hypermethylation was not seen for any of the three genes. Serum-free DNA was extracted and was tested for promoter hypermethylation. The status of serum-free DNA methylation was analyzed; the hypermethylation status was compared to clinical variables and cancer outcomes. Results: DNA hypermethylation was observed in 32% of samples for DAPK, 63% of samples for RARβP2 and 83% of samples for p16 from the cancer tissues. Among the 12 matched serum samples where the primary tumor showed hypermethylation in all three gene promoter regions, we were able to detect five incidences of serum DNA hypermethylation in four patients. The four patients had TNM stage II or higher disease. None of the patients with stage I disease showed serum-free DNA hypermethylation. Conclusion: Aberrant promoter hypermethylation was frequently observed in surgically resected adenocarcinoma of the lung. Concurrent serum-free DNA hypermethylation was detected in 34% of patients where the primary tumor showed hypermethylation in all three gene promoter regions. The findings suggest that the serum-free DNA methylation status might be used as a potential target for the diagnosis of lung cancer. However, the low sensitivity should be improved for use in a clinical application.

헬리코박터 파일로리 제균 치료가 위암 관련 유전자의 CpG Island 과염기화에 미치는 영향

최정민 ( Jeongmin Choi ),김상균 ( Sang Gyun Kim ),김병관 ( Byeong Gwan Kim ),고성준 ( Seong-joon Koh ),김지원 ( Ji Won Kim ),이국래 ( Kook Lae Lee ) 대한소화기학회 2016 대한소화기학회지 Vol.68 No.5

Background/Aims: Helicobacter pylori infection induces aberrant DNA methylation in gastric mucosa. We evaluated the long-term effect of H. pylori eradication on promotor CpG island hypermethylation in gastric carcinogenesis. Methods: H. pylori-positive patients with gastric adenoma or early gastric cancer who underwent endoscopic resection were enrolled. According to H. pylori eradication after endoscopic resection, the participants were randomly assigned to H. pylori eradication or non-eradication group. H. pylori-negative gastric mucosa from normal participants provided the normal control. CpG island hypermethylation of tumor-related genes (p16, CDH1, and RUNX-3) was evaluated by quantitative MethyLight assay in non-tumorous gastric mucosa. The gene methylation rate and median values of hypermethylation were compared after one year by H. pylori status. Results: In H. pylori-positive patients, hypermethylation of p16 was found in 80.6%, of CDH1 in 80.6%, and of RUNX-3 in 48.4%. This is significantly higher than normal control (p16, 10%; CDH1, 44%; RUNX-3, 16%) (p<0.05). In the H. pylori eradication group, methylation rates of p16 and CDH1 decreased in 58.1% and 61.3% of the patients, and the median values of hypermethylation were significantly lower at one year compared with the non-eradication group. However, RUNX-3 hypermethylation did not differ significantly at one year after H. pylori eradication. The non-eradication group hypermethylation did not change after one year. Conclusions: H. pylori infection was associated with promotor hypermethylation of genes in gastric carcinogenesis, and H. pylori eradication might reverse p16 and CDH1 hypermethylation. (Korean J Gastroenterol 2016;68:253-259)

5'-CpG Island Promoter Hypermethylation of the CAV-1 Gene in Breast Cancer Patients of Kashmir

Syeed, Nidda,Hussain, Firdous,Husain, Syed Akhtar,Siddiqi, Mushtaq A. Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.1

Background: Caveolin-1 (CAV-1), encoding the structural component of cellular caveolae, is a suggested tumor suppressor gene involved in cell signalling. Aberrant promoter methylation of CAV-1 is associated with inactivation of expression. We previously observed CAV-1 mutations in breast cancers and therefore devised this study to examine the hypermethylation status of the promoter region of CAV-1 with reference to breast cancer progression and development. Methods: Hypermethylation status of CAV-1 was analyzed by methylation specific PCR. Loss of expression of the CAV-1 gene was further evaluated by semi-quantitative rt-PCR. Results: 28/130 (21.5%) breast cancer cases showed promoter hypermethylation with reduced CAV-1 expression levels when compared with adjacent normal breast tissue. CAV-1 gene hypermethylation was significantly related to menopausal status, histopathological grade and age. Conclusion: The rationale of our study is that CAV-1 gene is transcriptionally repressed in breast cancer cells due to hypermethylation. Our results reveal that promoter hypermethylation and loss of expression of the CAV-1 gene is an important alternative mechanism for inactivation of CAV-1 leading to complete gene silencing.

The Overexpression of Histone Deacetylase 1 and Its Relationship with p16 INK4a Gene Hypermethylation in Pulmonary Squamous Cell Carcinoma and Adenocarcinoma

박종혁,최필조,홍영섭,김나영,이경은,노미숙 대한병리학회 2009 Journal of Pathology and Translational Medicine Vol.43 No.2

Background : DNA methylation and histone modification are dynamically linked in the epigenetic control of gene silencing and they play an important role in tumorigenesis. Methods : To evaluate the role of histone deacetylase 1 (HDAC1) in the development of lung cancer and the relationship between a HDAC1 overexpression and p16INK4a hypermethylation, we performed immunohistochemical staining for HDAC1 in 76 lung cancer specimens (39 squamous cell carcinomas and 37 adenocarcinomas) that had been previously evaluated for their p16INK4a methylation status by real-time quantitative polymerase chain reaction. Results : A HDAC1 overexpression (>50% of HDAC1 immunoreactive cells) was detected in 65 (85.5%) out of the 76 cases and it was more frequently seen in the squamous cell carcinomas (97.4%) than in the adenocarcinomas (73.0%) (p=0.002). The incidence of HDAC1 overexpression tended to be higher in the heavy smokers with more than 20 pack-years (p=0.067). Although there was no statistical significance, the frequency of p16INK4a hypermethylation in the cases with a HDAC1 overexpression (27.7%) tended to be higher than that in the cases without a HDAC1 overexpression (9.0%) (p=0.175). Conclusions : A HDAC1 overexpression might be involved in lung carcinogenesis, and especially in a subgroup of smoking and squamous cell carcinoma patients, and a HDAC1 overexpression may be associated with p16INK4a hypermethylation. Background : DNA methylation and histone modification are dynamically linked in the epigenetic control of gene silencing and they play an important role in tumorigenesis. Methods : To evaluate the role of histone deacetylase 1 (HDAC1) in the development of lung cancer and the relationship between a HDAC1 overexpression and p16INK4a hypermethylation, we performed immunohistochemical staining for HDAC1 in 76 lung cancer specimens (39 squamous cell carcinomas and 37 adenocarcinomas) that had been previously evaluated for their p16INK4a methylation status by real-time quantitative polymerase chain reaction. Results : A HDAC1 overexpression (>50% of HDAC1 immunoreactive cells) was detected in 65 (85.5%) out of the 76 cases and it was more frequently seen in the squamous cell carcinomas (97.4%) than in the adenocarcinomas (73.0%) (p=0.002). The incidence of HDAC1 overexpression tended to be higher in the heavy smokers with more than 20 pack-years (p=0.067). Although there was no statistical significance, the frequency of p16INK4a hypermethylation in the cases with a HDAC1 overexpression (27.7%) tended to be higher than that in the cases without a HDAC1 overexpression (9.0%) (p=0.175). Conclusions : A HDAC1 overexpression might be involved in lung carcinogenesis, and especially in a subgroup of smoking and squamous cell carcinoma patients, and a HDAC1 overexpression may be associated with p16INK4a hypermethylation.

BRD7 Promoter Hypermethylation as an Indicator of Well Differentiated Oral Squamous Cell Carcinomas

Balasubramanian, Anandh,Subramaniam, Ramkumar,Narayanan, Vivek,Annamalai, Thangavelu,Ramanathan, Arvind Asian Pacific Journal of Cancer Prevention 2015 Asian Pacific journal of cancer prevention Vol.16 No.4

Background: Promoter hypermethylation mediated gene silencing of tumor suppressor genes is considered as most frequent mechanism than genetic aberrations such as mutations in the development of cancers. BRD7 is a single bromodomain containing protein that functions as a subunit of SWI/SNF chromatin-remodeling complex to regulate transcription. It also interacts with the well know tumor suppressor protein p53 to trans-activate genes involved in cell cycle arrest. Loss of expression of BRD7 has been observed in breast cancers and nasopharyngeal carcinomas due to promoter hypermethylation. However, the genetic status of BRD7 in oral squamous cell carcinomas (OSCCs) is not known, although OSCC is one of the most common among all reported cancers in the Indian population. Hence, in the present study we investigated OSCC samples to determine the occurrence of hypermethylation in the promoter region of BRD7 and understand its prevalence. Materials and Methods: Genomic DNA extracted from biopsy tissues of twenty three oral squamous cell carcinomas were digested with methylation sensitive HpaII type2 restriction enzyme that recognizes and cuts unmethylated CCGG motifs. The digested DNA samples were amplified with primers flanking the CCGG motifs in promoter region of BRD7 gene. The PCR amplified products were analyzed by agarose gel electrophoresis along with undigested amplification control. Results: Methylation sensitive enzyme technique identified methylation of BRD7 promoter region seventeen out of twenty three (74%) well differentiated oral squamous cell carcinoma samples. Conclusions: The identification of BRD7 promoter hypermethylation in 74% of well differentiated oral squamous cell carcinomas indicates that the methylation dependent silencing of BRD7 gene is a frequent event in carcinogenesis. To the best of our knowledge, the present study is the first to report the occurrence of BRD7and its high prevalence in oral squamous cell carcinomas.

A Genome-Wide Methylation Approach Identifies a New Hypermethylated Gene Panel in Ulcerative Colitis

Kang, Keunsoo,Bae, Jin-Han,Han, Kyudong,Kim, Eun Soo,Kim, Tae-Oh,Yi, Joo Mi MDPI AG 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.8

<P>The cause of inflammatory bowel disease (IBD) is still unknown, but there is growing evidence that environmental factors such as epigenetic changes can contribute to the disease etiology. The aim of this study was to identify newly hypermethylated genes in ulcerative colitis (UC) using a genome-wide DNA methylation approach. Using an Infinium HumanMethylation450 BeadChip array, we screened the DNA methylation changes in three normal colon controls and eight UC patients. Using these methylation profiles, 48 probes associated with CpG promoter methylation showed differential hypermethylation between UC patients and normal controls. Technical validations for methylation analyses in a larger series of UC patients (<I>n</I> = 79) were performed by methylation-specific PCR (MSP) and bisulfite sequencing analysis. We finally found that three genes (<I>FAM217B</I>, <I>KIAA1614</I> and <I>RIBC2</I>) that were significantly elevating the promoter methylation levels in UC compared to normal controls. Interestingly, we confirmed that three genes were transcriptionally silenced in UC patient samples by qRT-PCR, suggesting that their silencing is correlated with the promoter hypermethylation. Pathway analyses were performed using GO and KEGG databases with differentially hypermethylated genes in UC. Our results highlight that aberrant hypermethylation was identified in UC patients which can be a potential biomarker for detecting UC. Moreover, pathway-enriched hypermethylated genes are possibly implicating important cellular function in the pathogenesis of UC. Overall, this study describes a newly hypermethylated gene panel in UC patients and provides new clinical information that can be used for the diagnosis and therapeutic treatment of IBD.</P>

Promoter Methylation Status of DNA Repair Gene (hMLH1) in Gastric Carcinoma Patients of the Kashmir Valley

Wani, Majid,Afroze, Dil,Makhdoomi, Muzamil,Hamid, Iqra,Wani, Bilal,Bhat, Gulzar,Wani, Rauf,Wani, Khursheed Asian Pacific Journal of Cancer Prevention 2012 Asian Pacific journal of cancer prevention Vol.13 No.8

Cancer is a multi-factorial disease and variation in genetic susceptibility, due to inherited differences in the capacity to repair mismatches in the genome, is an important factor in the development of gastric cancer (GC), for example. Epigenetic changes, including aberrant methylation of 5/CpG islands in the promoter regions of mismatch repair (MMR) genes like hMLH1, have been implicated in the development of various types of GC. In the present study we evaluated the role of hMLH1 promoter hypermethylation in Kashmiri GC patients and controls, and assessed correlations with various dietary and lifestyle factors. The study included 70 GC patients (56 males and 14 females; age ($mean{\pm}S.D$) $50{\pm}11.4$ years). Distinction between methylated and unmethylated was achieved with MS-PCR and DNA band patterns. The Chi-square test was applied to assess the risk due to promoter hypermethylation. We found a strikingly high frequency of promoter hypermethylation in GC cases than in normal samples (72.9% (51/70) in GC cases vs 20% (14/70) in normal samples (p=0.0001).We also observed a statistically significant association between methylated hMLH1 gene promoter and smoking, consumption of sundried vegetables and hot salted tea with the risk of GC. This study revealed that hMLH1 hypermethylation is strongly associated with GC and suggested roles for epigenetic changes in stomach cancer causation in the Kashmir valley.

대장암에서 관찰되는 Caveolin 의 낮은 발현과 프로모터 CpG 과메틸화에 대한 연구

김남훈 ( Nam Hoon Kim ),김효종 ( Hyo Jong Kim ),지성길 ( Sung Gil Chi ),문영수 ( Young Soo Moon ) 대한장연구학회 2007 Intestinal Research Vol.5 No.1

Background/Aims: Abnormal reduction of caveolins has been found in many human cancers while its overexpression also correlates with increased metastatic progression of some tumors. To elucidate the possible implication of caveolin abnormality in human colon tumorigenesis, the expression and mutational status of caveolins was explored. Methods: We investigated 11 human colon cancer cell lines, 49 primary carcinoma tissues, and its matched normal colonic tissues. Both mRNA and protein levels of caveolins (cav-1, cav-2) were evaluated by quantitative RT-PCR and immunoblotting. Effect of cav-1 expression on tumor growth was tested using cell counting and colony formation assay. Cav-1 expression was restored in nonexpressing cells, whereas cav-1 expression was inhibited by siRNA-mediated knockdown in expressing cells. Methylation status of 38 CpG sites was evaluated by bisulfite DNA sequencing. Results: Low expression of cav-1 transcript was found in 54.5% of cancer cell lines, whereas 45.5% of those showed strong expression. Expression level of cav-1 protein was very low in majority of cancer cell lines except two cell lines. Approximately 47% and 10% of primary carcinomas exhibited significant reduction and elevation in cav-1 expression, respectively. Cav-2 expression also showed down- and up-regulation in 28% and 3% of primary tumors, respectively. Cav-1 transcript was re-expressed in nonexpressing cells by 5-aza-dC treatment. Restoration of cav-1 inhibited growth of cav-1-negative cells and reduced phospho-Erk level, whereas ectopic overexpression of cav-1 further stimulated cav-1-expressing cells and activated p53 and p21. Conclusions: Caveolin undergoes epigenetic silencing in a considerable proportion of human colon cancers by aberrant promoter CpG hypermethylation. Also, cav-1 acts two opposite functions as a growth suppressor or growth stimulator in colon cancers. (Intest Res 2007;5:60-72)

Hypermethylation of the <i>COX-2</i> gene is a potential prognostic marker for cervical cancer

Jo, Hoenil,Kang, Sokbom,Kim, Jae W.,Kang, Gyeong H.,Park, Noh H.,Song, Yong S.,Park, Sang Y.,Kang, Soon B.,Lee, Hyo P. unknown 2007 Journal of Obstetrics and Gynaecology Research Vol. No.

<P>Abstract</P><P>Aim: </P><P>The aim of the present study was to evaluate the DNA hypermethylation profiles of 14 genes known to be associated with tumor behavior and their clinical significance in cervical cancer.</P><P>Method: </P><P>The clinical features of 82 patients with stage IB cervical cancer were analyzed in terms of DNA hypermethylation of 14 genes (<I>hMLH1</I>, <I>p16</I>, <I>COX-2</I>, <I>CDH1</I>, <I>APC</I>, <I>DAPK</I>, <I>MGMT</I>, <I>p14</I>, <I>RASSF1A</I>, <I>RUNX3</I>, <I>TIMP3</I>, <I>FHIT</I>, <I>THBS1</I>, and <I>HLTF</I>).</P><P>Results: </P><P>Of 14 genes investigated, only hypermethylation of <I>COX-2</I> showed significant association with poor disease-free survival (<I>P</I> = 0.001). To further investigate an alteration in <I>COX-2</I> expression by DNA hypermethylation, immunohistochemistry for <I>COX-2</I> protein was performed in the cervical cancer tissues. We found no significant association between hypermethylation and expression patterns of the <I>COX-2</I> gene.</P><P>Conclusions: </P><P>The present results suggest that DNA hypermethylation of the <I>COX-2</I> gene may be a potential prognostic marker in early stage cervical cancer, the underlying mechanism of which is independent of gene silencing.</P>

Quantitative Analysis of Cancer-associated Gene Methylation Connected to Risk Factors in Korean Colorectal Cancer Patients

Kang, Ho-Jin,Kim, Eun-Jeong,Kim, Byoung-Gwon,You, Chang-Hun,Lee, Sang-Yong,Kim, Dong-Il,Hong, Young-Seoub The Korean Society for Preventive Medicine 2012 Journal of Preventive Medicine and Public Health Vol.45 No.4

Objectives: The purpose of this paper was to elucidate the potential methylation levels of adjacent normal and cancer tissues by comparing them with normal colorectal tissues, and to describe the correlations between the methylation and clinical parameters in Korean colorectal cancer (CRC) patients. Methods: Hypermethylation profiles of nine genes (RASSF1, APC, $p16^{INK4a}$, Twist1, E-cadherin, TIMP3, Smad4, COX2, and ABCB1) were examined with 100 sets of cancer tissues and 14 normal colorectal tissues. We determined the hypermethylation at a given level by a percent of methylation ratio value of 10 using quantitative methylation real-time polymerase chain reaction. Results: Nine genes' hypermethylation levels in Korean CRC patient tissues were increased more higher than normal colorectal tissues. However, the amounts of $p16^{INK4a}$ and E-cadherin gene hypermethylation in normal and CRC tissues were not significantly different nor did TIMP3 gene hypermethylation in adjacent normal and cancer tissues differ significantly. The hypermethylation of TIMP3, Ecadherin, ABCB1, and COX2 genes among other genes were abundantly found in normal colorectal tissues. The hypermethylation of nine genes' methylation in cancer tissues was not significantly associated with any clinical parameters. In Cohen's kappa test, it was moderately observed that RASSF1 was related with E-cadherin, and Smad4 with ABCB1 and COX2. Conclusions: This study provides evidence for different hypermethylation patterns of cancer-associated genes in normal and CRC tissues, which may serve as useful information on CRC cancer progression.