Epigenetic Regulation of the Tumour Suppressor RASSF1A in Bone Cancer Cells: DNA Methylation Study

Objective: Osteosarcoma is a bone cancer that affects children and adolescents. The RASSF1A is a tumor suppressor capable of mediating the regulation of cell cycle arrest, migration, including apoptosis. It is the most continually silenced gene that contributes to human cancer. Furthermore, RASSF1A functions as a scaffold protein that can regulate microtubules network and bind apoptotic kinases MST1 and MST2 via the Sav-RASSF-Hippo domain. Epigenetic inactivation of genes by DNA methylation is a key factor regulating gene expression and genomic stability. Our aim was to study the RASSF1A gene promoter methylation in three osteosarcomas (U2OS, Saos-2, and MG-63), two Ewing Sarcoma (A-673 and SK-ES-1), and one-fibrosarcoma (HT-1080) cell lines. Materials and Methods: Three osteosarcomas (U2OS, Saos-2, and MG-63), two Ewing Sarcoma (A-673 and SK-ES-1), and one-fibrosarcoma (HT-1080) cell lines were used to study RASSF1A gene promoter methylation, using bisulphite conversion of DNA, followed by methylation-specific polymerase chain reaction (PCR) Results: The RASSFIA’s gene promoter methylation was established as a frequent event. Hypermethylation of RASSF1A promoter, was detected in five out of six studied cell lines. Conclusions: These results demonstrated that altering the Sav-RASSF1-Hippo may be accomplished through hypermethylation of RASSF1A and may play an essential role in Ewing’s sarcoma and Osteosarcoma. The methylation pattern of Sav-RASSF1-Hippo tumor suppressor pathway in human bone cancer along with RASSF1A expression with its effector proteins merits further investigation. This may reveal how the RASSFIA has a physiological signal transduction, including how the process of its deregulation can contribute to transformation of the cell, eventually leading to the incorporation of novel therapeutic options with improved prognosis for bone cancer.

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DNA methylation profiling in MEN1-related pancreatic neuroendocrine tumors reveals a potential epigenetic target for treatment

Acta Endocrinologica ◽

10.1530/eje-18-0195 ◽

2018 ◽

Vol 179 (3) ◽

pp. 153-160 ◽

Cited By ~ 5

Author(s):

E B Conemans ◽

L Lodewijk ◽

C B Moelans ◽

G J A Offerhaus ◽

C R C Pieterman ◽

...

Keyword(s):

Dna Methylation ◽

Tumor Suppressor ◽

Promoter Methylation ◽

Tumor Suppressor Genes ◽

Gene Promoter ◽

Pancreatic Neuroendocrine Tumor ◽

Promoter Hypermethylation ◽

Suppressor Gene ◽

Suppressor Genes ◽

Frequent Event

ObjectiveEpigenetic changes contribute to pancreatic neuroendocrine tumor (PanNET) development. Hypermethylation of promoter DNA as a cause of tumor suppressor gene silencing is a well-established oncogenic mechanism that is potentially reversible and therefore an interesting therapeutic target. Multiple endocrine neoplasia type 1 (MEN1) is the most frequent cause of inherited PanNETs. The aim of this study was to determine promoter methylation profiles in MEN1-related PanNETs.Design and methodsMethylation-specific multiplex ligation-dependent probe amplification was used to assess promoter methylation of 56 tumor suppressor genes in MEN1-related (n = 61) and sporadic (n = 34) PanNETs. Differences in cumulative methylation index (CMI), individual methylation percentages and frequency of promoter hypermethylation between subgroups were analyzed.ResultsWe found promoter methylation of a large number of potential tumor suppressor genes. CMI (median CMI: 912 vs 876,P = 0.207) was the same in MEN1-related and sporadic PanNETs. We found higher methylation percentages ofCASP8in MEN1-related PanNETs (median: 59% vs 16.5%,P = 0.002). In MEN1-related non-functioning PanNETs, the CMI was higher in larger PanNETs (>2 cm) (median: 969.5 vs 838.5;P = 0.021) and in PanNETs with liver metastases (median: 1036 vs 869;P = 0.013). Hypermethylation ofMGMT2was more frequent in non-functioning PanNETs compared to insulinomas (median: 44.7% vs 8.3%;P = 0.022). Hypermethylation of the Von Hippel–Lindau gene promoter was observed in one MEN1-related PanNET and was associated with loss of protein expression.ConclusionPromoter hypermethylation is a frequent event in MEN1-related and sporadic PanNETs. Targeting DNA methylation could be of therapeutic value in MEN1 patients with advanced PanNETs.

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Global DNA methylation and tumor suppressor gene promoter methylation and gastric cancer risk in an Omani Arab population

Epigenomics ◽

10.2217/epi.11.65 ◽

2011 ◽

Vol 3 (4) ◽

pp. 417-429 ◽

Cited By ~ 7

Author(s):

Jennifer A Rusiecki ◽

Maryam Al-Nabhani ◽

Letizia Tarantini ◽

Ligong Chen ◽

Andrea Baccarelli ◽

...

Keyword(s):

Gastric Cancer ◽

Dna Methylation ◽

Tumor Suppressor ◽

Cancer Risk ◽

Promoter Methylation ◽

Gene Promoter ◽

Suppressor Gene ◽

Global Dna Methylation ◽

Gastric Cancer Risk ◽

Arab Population

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SCT promoter methylation is a highly discriminative biomarker for lung and many other cancers

10.1101/018515 ◽

2015 ◽

Author(s):

Adwait Sathe ◽

Xiaotu Ma ◽

Michael Zhang

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Promoter Methylation ◽

Cpg Island ◽

Gene Promoter ◽

Nsclc Cell ◽

Primary Tumors ◽

Lung Cancers ◽

Cancer Types ◽

Aberrant Dna Methylation

Aberrant DNA methylation has long been implicated in cancers. In this work we present a highly discriminative DNA methylation biomarker for non-small cell lung cancers and fourteen other cancers. Based on 69 NSCLC cell lines and 257 cancer-free lung tissues we identified a CpG island in SCT gene promoter which was verified by qMSP experiment in 15 NSCLC cell lines and 3 immortalized human respiratory epithelium cells. In addition, we found that SCT promoter was methylated in 23 cancer cell lines involving >10 cancer types profiled by ENCODE. We found that SCT promoter is hyper-methylated in primary tumors from TCGA lung cancer cohort. Additionally, we found that SCT promoter is methylated at high frequencies in fifteen malignancies and is not methylated in ~1000 non-cancerous tissues across >30 organ types. Our study indicates that SCT promoter methylation is a highly discriminative biomarker for lung and many other cancers.

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Efficacy of decitabine in malignant meningioma cells: relation to promoter demethylation of distinct tumor suppressor and oncogenes and independence from TERT

Journal of Neurosurgery ◽

10.3171/2020.7.jns193097 ◽

2020 ◽

pp. 1-10

Author(s):

Louise Stögbauer ◽

Christian Thomas ◽

Andrea Wagner ◽

Nils Warneke ◽

Eva Christine Bunk ◽

...

Keyword(s):

Dna Methylation ◽

Tumor Suppressor ◽

Promoter Methylation ◽

Tumor Suppressor Genes ◽

Telomerase Activity ◽

High Grade ◽

Htert Promoter ◽

Men 1 ◽

Meningioma Cell ◽

Tert Expression

OBJECTIVEChemotherapeutic options for meningiomas refractory to surgery or irradiation are largely unknown. Human telomerase reverse transcriptase (hTERT) promoter methylation with subsequent TERT expression and telomerase activity, key features in oncogenesis, are found in most high-grade meningiomas. Therefore, the authors investigated the impact of the demethylating agent decitabine (5-aza-2ʹ-deoxycytidine) on survival and DNA methylation in meningioma cells.METHODShTERT promoter methylation, telomerase activity, TERT expression, and cell viability and proliferation were investigated prior to and after incubation with decitabine in two benign (HBL-52 and Ben-Men 1) and one malignant (IOMM-Lee) meningioma cell line. The global effects of decitabine on DNA methylation were additionally explored with DNA methylation profiling.RESULTSHigh levels of TERT expression, telomerase activity, and hTERT promoter methylation were found in IOMM-Lee and Ben-Men 1 but not in HBL-52 cells. Decitabine induced a dose-dependent significant decrease of proliferation and viability after incubation with doses from 1 to 10 μM in IOMM-Lee but not in HBL-52 or Ben-Men 1 cells. However, effects in IOMM-Lee cells were not related to TERT expression, telomerase activity, or hTERT promoter methylation. Genome-wide methylation analyses revealed distinct demethylation of 14 DNA regions after drug administration in the decitabine-sensitive IOMM-Lee but not in the decitabine-resistant HBL-52 cells. Differentially methylated regions covered promoter regions of 11 genes, including several oncogenes and tumor suppressor genes that to the authors’ knowledge have not yet been described in meningiomas.CONCLUSIONSDecitabine decreases proliferation and viability in high-grade but not in benign meningioma cell lines. The effects of decitabine are TERT independent but related to DNA methylation changes of promoters of distinct tumor suppressor genes and oncogenes.

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Genome-wide analysis identifies critical DNA methylations within NTRKs genes in colorectal cancer

Journal of Translational Medicine ◽

10.1186/s12967-021-02740-6 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Zijian Chen ◽

Zenghong Huang ◽

Yanxin Luo ◽

Qi Zou ◽

Liangliang Bai ◽

...

Keyword(s):

Colorectal Cancer ◽

Dna Methylation ◽

Promoter Methylation ◽

Expression Profiles ◽

Methylation Status ◽

Gene Promoter ◽

Normal Mucosa ◽

Suppressor Gene ◽

Survival Outcome ◽

Prognostic Models

Abstract Background Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the methylation and expression profiles and prognostic value of NTRKs gene in colorectal cancer (CRC). Methods An analysis of DNA methylation and expression profiles in CRC patients was performed to explore the critical methylations within NTRKs genes. The methylation marker was validated in a retrospectively collected cohort of 229 CRC patients and tested in other tumor types from TCGA. DNA methylation status was determined by quantitative methylation-specific PCR (QMSP). Results The profiles in six CRC cohorts showed that NTRKs gene promoter was more frequently methylated in CRC compared to normal mucosa, which was associated with suppressed gene expression. We identified a specific methylated region within NTRK3 promoter targeted by cg27034819 and cg11525479 that best predicted survival outcome in CRC. NTRK3 promoter methylation showed independently predictive value for survival outcome in the validation cohort (P = 0.004, HR 2.688, 95% CI [1.355, 5.333]). Based on this, a nomogram predicting survival outcome was developed with a C-index of 0.705. Furthermore, the addition of NTRK3 promoter methylation improved the performance of currently-used prognostic model (AIC: 516.49 vs 513.91; LR: 39.06 vs 43.64, P = 0.032). Finally, NTRK3 promoter methylation also predicted survival in other tumors, including pancreatic cancer, glioblastoma and stomach adenocarcinoma. Conclusions This study highlights the essential value of NTRK3 methylation in prognostic evaluation and the potential to improve current prognostic models in CRC and other tumors.

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Analysis of smad4 gene promoter methylation in pancreatic and endometrial cancers

Archive of oncology ◽

10.2298/aoo1702017n ◽

2017 ◽

Vol 23 (2) ◽

pp. 17-19

Author(s):

Aleksandra Nikolic ◽

Filip Opincal ◽

Momcilo Ristanovic ◽

Jovanka Trifunovic ◽

Srbislav Knezevic ◽

...

Keyword(s):

Promoter Methylation ◽

Methylation Status ◽

Gene Promoter ◽

Promoter Hypermethylation ◽

Surgical Removal ◽

Smad4 Gene ◽

Frequent Event ◽

Cancer Types ◽

Endometrial Cancers ◽

Tumor Types

Background. Promoter hypermethylation of the SMAD4 gene has been registered in some cancer types, but in general doesn?t appear to be a frequent event in carcinogenesis. However, only a few published studies deal with this topic and not many cancer types have been analyzed. The aim of this study was to establish SMAD4 gene promoter methylation status in pancreatic and endometrial cancers. Methods. Patients included in the study (62 subjects) were diagnosed and surgically treated at the University of Belgrade, Clinical Center of Serbia. Patients with pancreatic carcinoma (17 subjects) underwent surgical removal of the pancreatic adenocarcinoma at the First Surgical Clinic, while the patients with endometrial carcinoma (45 subjects) underwent hysterectomy with adnexectomy at the Institute for Gynecology and Obstetrics. Extraction of DNA from fresh tissue samples was performed and the methylation status of the SMAD4 gene promoter was studied by a previously designed PCR-based HpaII and MspI restriction enzyme assay. The resulting PCR products were analyzed by electrophoresis in 2% agarose gels. Results. Neither of the analyzed samples was found to be hypermethylated. Conclusion. This is the first report on SMAD4 methylation status in pancreatic and endometrial tumor specimens, and supports the viewpoint that SMAD4 hypermethylation is not a common event in malignant tumors. Nevertheless, promoter hypermethylation remains a candidate mechanism for SMAD4 inactivation in malignant tissue as a potential cause of decreased or lost SMAD4 expression in certain tumor types, and should be further investigated in different tumor types and larger cohorts of patients.

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Enhanced CXCR4 Expression Associates with Increased Gene Body 5-Hydroxymethylcytosine Modification but not Decreased Promoter Methylation in Colorectal Cancer

Cancers ◽

10.3390/cancers12030539 ◽

2020 ◽

Vol 12 (3) ◽

pp. 539 ◽

Cited By ~ 1

Author(s):

Alexei J. Stuckel ◽

Wei Zhang ◽

Xu Zhang ◽

Shuai Zeng ◽

Urszula Dougherty ◽

...

Keyword(s):

Colorectal Cancer ◽

Dna Methylation ◽

Cell Lines ◽

Promoter Methylation ◽

Cpg Island ◽

Methylation Status ◽

Cxcr4 Expression ◽

Normal Colonic Mucosa ◽

Gene Body ◽

Expression Levels

In colorectal cancer (CRC), upregulation of the C-X-C motif chemokine receptor 4 (CXCR4) is correlated with metastasis and poor prognosis, highlighting the need to further elucidate CXCR4’s regulation in CRC. For the first time, DNA methylation and 5-hydroxymethylcytosine aberrations were investigated to better understand the epigenetic regulation of CXCR4 in CRC. CXCR4 expression levels were measured using qPCR and immunoblotting in normal colon tissues, primary colon cancer tissues and CRC cell lines. Publicly available RNA-seq and methylation data from The Cancer Genome Atlas (TCGA) were extracted from tumors from CRC patients. The DNA methylation status spanning CXCR4 gene was evaluated using combined bisulfite restriction analysis (COBRA). The methylation status in the CXCR4 gene body was analyzed using previously performed nano-hmC-seal data from colon cancers and adjacent normal colonic mucosa. CXCR4 expression levels were significantly increased in tumor stromal cells and in tumor colonocytes, compared to matched cell types from adjacent normal-appearing mucosa. CXCR4 promoter methylation was detected in a minority of colorectal tumors in the TCGA. The CpG island of the CXCR4 promoter showed increased methylation in three of four CRC cell lines. CXCR4 protein expression differences were also notable between microsatellite stable (MSS) and microsatellite instable (MSI) tumor cell lines. While differential methylation was not detected in CXCR4, enrichment of 5-hydroxymethylcytosine (5hmC) in CXCR4 gene bodies in CRC was observed compared to adjacent mucosa.

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Thrombomodulin Is Silenced in Malignant Mesothelioma by a Poly(ADP-ribose) Polymerase-1-mediated Epigenetic Mechanism

Journal of Biological Chemistry ◽

10.1074/jbc.m110.217331 ◽

2011 ◽

Vol 286 (22) ◽

pp. 19478-19488 ◽

Cited By ~ 18

Author(s):

Linda Nocchi ◽

Marco Tomasetti ◽

Monica Amati ◽

Jiri Neuzil ◽

Lory Santarelli ◽

...

Keyword(s):

Dna Methylation ◽

Malignant Mesothelioma ◽

Promoter Methylation ◽

Dna Methyltransferase ◽

Critical Role ◽

Gene Promoter ◽

Epigenetic Mechanism ◽

Heterogeneous Expression ◽

High Level

Malignant mesothelioma (MM) is often complicated by thromboembolic episodes, with thrombomodulin (TM) playing a critical role in the anticoagulant process. Heterogeneous expression of TM has been observed in cancer, and low or no TM expression in cancer cells is associated with poor prognosis. In this study, we analyzed TM expression in biopsies of MM patients and compared them with normal mesothelial tissue. The role of DNA methylation-associated gene silencing in TM expression was investigated. To evaluate poly(ADP-ribose) polymerase-1 (PARP1) as responsible for gene promoter epigenetic modifications, nonmalignant mesothelial cells (Met-5A) and MM cells (H28) were silenced for PARP1 and the DNA methylation/acetylation-associated TM expression evaluated. A correlation between low TM expression and high level of TM promoter methylation was found in MM biopsies. Low expression of TM was restored in MM cells by their treatment with 5-aza-2′-deoxycytidine and, to a lesser extent, with trichostatin, whereas the epigenetic agents did not affect TM expression in Met-5A cells. Silencing of PARP1 resulted in a strong down-regulation of TM expression in Met-5A cells, while restoring TM expression in H28 cells. PARP1 silencing induced TM promoter methylation in Met-5A cells and demethylation in MM cells, and this was paralleled by corresponding changes in the DNA methyltransferase activity. We propose that methylation of the TM promoter is responsible for silencing of TM expression in MM tissue, a process that is regulated by PARP1.

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