Undraga Schagdarsurengin

Frequent RASSF1A promoter hypermethylation and K-ras mutations in pancreatic carcinoma

Recently, we have characterized the Ras association domain family 1A gene (RASSF1A) at the segment 3p21.3, which is frequently lost in variety of human cancers and epigenetically inactivated in many types of primary tumors, such as lung, breast, kidney, prostate and thyroid carcinomas. Here, we investigated the methylation status of the RASSF1A CpG island promoter in the pathogenesis of pancreatic cancer. RASSF1A hypermethylation was detected in 29 out of 45 (64%) primary adenocarcinomas, in 10 out of 12 (83%) endocrine tumors and in eight out of 18 (44%) pancreatitis samples. In seven out of eight pancreas cancer cell lines, RASSF1A was silenced and was retranscribed after treatment with 5-aza-2′-deoxycytidine. Additionally, we analysed the aberrant methylation frequency of cell cycle inhibitor p16INK4a and K-ras gene mutations in the pancreatic samples. p16 inactivation was detected in 43% of adenocarcinomas, in 17% of neuroendocrine tumors, in 18% of pancreatitis and in 63% of pancreas cancer cell lines. K-ras mutations were detected in 16 out of 45 (36%) primary adenocarcinomas. Pancreatic adenocarcinomas with K-ras mutation have significantly less RASSF1A methylation and vice versa (P=0.001, χ2 test). In conclusion, our data indicate that inactivation of the RASSF1A gene is a frequent event in pancreatic cancer and suggest an inverse correlation between RASSF1A silencing and K-ras activation.

Frequent epigenetic inactivation of the RASSF1A gene in hepatocellular carcinoma

Aberrant promoter methylation is a fundamental mechanism of inactivation of tumor suppressor genes in cancer. The Ras association domain family 1A gene (RASSF1A) is frequently epigenetically silenced in several types of human solid tumors. In this study, we have investigated the expression and methylation status of the RASSF1A gene in hepatocellular carcinoma (HCC). In two HCC cell lines (HepG2 and Hep3B) RASSF1A was inactivated and treatment of these cell lines with a DNA methylation inhibitor reactivated the transcription of RASSF1A. The methylation status of the RASSF1A promoter region was analysed in 26 primary liver tissues including HCC, hepatocellular adenoma (HCA), liver fibrosis, hepatocirrhosis. Out of 15, 14 (93%) HCC were methylated at the RASSF1A CpG island and hypermethylation was independent of hepatitis virus infection. RASSF1A was also methylated in two out of two fibrosis and in three (75%) out of four cirrhosis; the latter carries an increased risk of developing HCC. Additionally, we analysed the methylation status of p16INK4a and other cancer-related genes in the same liver tumors. Aberrant methylation in the HCC samples was detected in 71% of samples for p16, 25% for TIMP3, 17% for PTEN, 13% for CDH1, and 7% for RARβ2. In conclusion, our results demonstrate that RASSF1A and p16INK4a inactivation by methylation are frequent events in hepatocellular carcinoma, but not in HCA, which is in contrast to HCC without cirrhosis, viral hepatitis, storage diseases, or genetic background. Therefore, this study gives additional evidence against a progression of adenoma to carcinoma in the liver. Thus, RASSF1A hypermethylation could be useful as a marker of malignancy and to distinguish between the distinct forms of highly differentiated liver neoplasm.

Chromatin Inactivation Precedes De Novo DNA Methylation during the Progressive Epigenetic Silencing of the RASSF1A Promoter

ABSTRACT Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.

Frequent hypermethylation of MST1 and MST2 in soft tissue sarcoma

The RASSF1A tumor suppressor is involved in regulation of apoptosis and cell cycle progression. RASSF1A is localized to microtubules and binds the apoptotic kinases MST1 and MST2. It has been shown that this interaction is mediated by the Sav‐RASSF‐Hpo domain, which is an interaction domain characterized for the Drosophila proteins Sav (human WW45), Hpo (human MST1 and MST2) and Warts/LATS (large tumor suppressor). Previously, we have reported that RASSF1A hypermethylation occurs frequently in soft tissue sarcoma and is associated with an unfavorable prognosis for cancer patients. In our study, we performed methylation analysis of the CpG island promoter of MST1, MST2, WW45, LATS1 and LATS2 in soft tissue sarcomas by methylation‐specific PCR. No or a very low methylation frequency was detected for WW45, LATS1 and LATS2 (<7%). In 19 out of 52 (37%) sarcomas, a methylated promoter of MST1 was detected and 12 out of 60 (20%) samples showed methylation of the MST2 promoter. Methylation status of MST1 was confirmed by bisulfite sequencing. In tumors harboring a methylated promoter of MST1, a reduction of MST1 expression was observed by RT‐PCR. In leiomyosarcomas, MST1 and MST2 or RASSF1A methylation were mutually exclusive (P = 0.007 and P = 0.025, respectively). Surprisingly, a significantly increased risk for tumor‐related death was found for patients with an unmethylated MST1 promoter (P = 0.036). In summary, our results suggest that alteration of the Sav‐RASSF1‐Hpo tumor suppressor pathway may occur through hypermethylation of the CpG island promoter of MST1, MST2 and/or RASSF1A in human sarcomas.

Analysing the sperm epigenome: roles in early embryogenesis and assisted reproduction.

An understanding of the epigenetic mechanisms involved in sperm production and their impact on the differentiating embryo is essential if we are to optimize fertilization and assisted reproduction techniques in the future. Male germ cells are unique in terms of size, robustness, and chromatin structure, which is highly condensed owing to the replacement of most histones by protamines. Analysis of sperm epigenetics requires specific techniques that enable the isolation of high quality chromatin and associated nucleic acids. Histone modification, DNA methylation and noncoding RNAs have important, but so far underestimated, roles in the production of fertile sperm. Aberrations in these epigenetic processes have detrimental consequences for both early embryo development and assisted reproductive technology. Emerging computational techniques are likely to improve our understanding of chromatin dynamics in the future.

Epigenetics in male reproduction: effect of paternal diet on sperm quality and offspring health

Epigenetic inheritance and its underlying molecular mechanisms are among the most intriguing areas of current biological and medical research. To date, studies have shown that both female and male germline development follow distinct paths of epigenetic events and both oocyte and sperm possess their own unique epigenomes. Fertilizing male and female germ cells deliver not only their haploid genomes but also their epigenomes, which contain the code for preimplantation and postimplantation reprogramming and embryonal development. For example, in spermatozoa, DNA methylation profile, DNA-associated proteins, protamine 1:protamine 2 ratio, nucleosome distribution pattern, histone modifications and other properties make up a unique epigenetic landscape. However, epigenetic factors and mechanisms possess certain plasticity and are affected by environmental conditions. Paternal and maternal lifestyle, including physical activity, nutrition and exposure to hazardous substances, can alter the epigenome and, moreover, can affect the health of their children. In male reproductive health, data are emerging on epigenetically mediated effects of a mans diet on sperm quality, for example through phytochemicals, minerals and vitamins, and nutritional support for subfertile men is already being used. In addition, studies in animal models and human epidemiological data point toward a transgenerational effect of the paternally contributed sperm epigenome on offspring health.

Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences

BackgroundThe Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 and the effectors MST1, MST2 and WW45 in thyroid carcinogenesis.ResultsFrequent methylation of the RASSF2 and RASSF5A CpG island promoters in thyroid tumors was observed. RASSF2 was methylated in 88% of thyroid cancer cell lines and in 63% of primary thyroid carcinomas. RASSF2 methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid, goiter and follicular adenoma (0%, 17% and 0%, respectively; p < 0.05). Patients which were older than 60 years were significantly hypermethylated for RASSF2 in their primary thyroid tumors compared to those younger than 40 years (90% vs. 38%; p < 0.05). RASSF2 promoter hypermethylation correlated with its reduced expression and treatment with a DNA methylation inhibitor reactivated RASSF2 transcription. Over-expression of RASSF2 reduced colony formation of thyroid cancer cells. Functionally our data show that RASSF2 interacts with the proapoptotic kinases MST1 and MST2 and induces apoptosis in thyroid cancer cell lines. Deletion of the MST interaction domain of RASSF2 reduced apoptosis significantly (p < 0.05).ConclusionThese results suggest that RASSF2 encodes a novel epigenetically inactivated candidate tumor suppressor gene in thyroid carcinogenesis.

Frequent epigenetic inactivation of RASSF10 in thyroid cancer

The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of a novel RASSF member termed RASSF10 in thyroid carcinogenesis. The RASSF10 CpG island promoter was intensively methylated in nine thyroid cancer cell lines and in 66% of primary thyroid carcinomas. RASSF10 methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid and follicular adenoma (0% and 10%, respectively; p

Frequent epigenetic inactivation of cystatin M in breast carcinoma.

Cystatin M is a potent endogenous inhibitor of lysosomal cysteine proteases. In breast carcinoma, cystatin M expression is frequently downregulated. It has been shown that cystatin M expression suppressed growth and migration of breast cancer cells. We examined the methylation status of the CpG island promoter of cystatin M in four breast cancer cell lines (MDAMB231, ZR75-1, MCF7 and T47D), in 40 primary breast carcinoma and in corresponding normal tissue probes by combined bisulphite restriction analysis. To investigate the effects of cystatin M expression on the growth of breast carcinoma, cystatin M was transfected in T47D. The cystatin M promoter was highly methylated in all four-breast cancer cell lines. Primary breast tumours were significantly more frequently methylated compared to normal tissue samples (60 vs 25%; P=0.006 Fishers exact test). Treatment of breast cancer cells with 5-aza-2′-deoxycytidine (5-Aza-CdR), reactivated the transcription of cystatin M. Transfection of breast carcinoma cells with cystatin M caused a 30% decrease in colony formation compared to control transfection (P=0.002). Our results show that cystatin M is frequently epigenetically inactivated during breast carcinogenesis and cystatin M expression suppresses the growth of breast carcinoma. These data suggest that cystatin M may encode a novel epigenetically inactivated candidate tumour suppressor gene.

Promoter methylation and loss of coding exons of the fragile histidine triad (FHIT) gene in intrahepatic cholangiocarcinomas

Abstract: Aims: About 10–30% of primary liver cancers represent intrahepatic cholangiocarcinomas (IHCC). Since chromosomal losses of 3p are detectable in about 40% of cholangiocarcinomas our study aimed at the identification of mechanisms leading to functional deletion of tumor suppressor genes in this region. Our efforts focussed on genomic losses and epigenetic inactivation of two tumor suppressor genes, the fragile histidine triad (FHIT) and the ras association domain family 1 (RASSF1A) genes, both located on the short arm of chromosome 3.