The RASSF gene family members RASSF5, RASSF6 and RASSF7 show frequent DNA methylation in neuroblastoma

Molecular Cancer (Impact Factor: 4.26). 06/2012; 11(1):40. DOI: 10.1186/1476-4598-11-40
Source: PubMed


Hypermethylation of promotor CpG islands is a common mechanism that inactivates tumor suppressor genes in cancer. Genes belonging to the RASSF gene family have frequently been reported as epigenetically silenced by promotor methylation in human cancers. Two members of this gene family, RASSF1A and RASSF5A have been reported as methylated in neuroblastoma. Data from our previously performed genome-wide DNA methylation array analysis indicated that other members of the RASSF gene family are targeted by DNA methylation in neuroblastoma.

In the current study, we found that several of the RASSF family genes (RASSF2, RASSF4, RASSF5, RASSF6, RASSF7, and RASSF10) to various degrees were methylated in neuroblastoma cell lines and primary tumors. In addition, several of the RASSF family genes showed low or absent mRNA expression in neuroblastoma cell lines. RASSF5 and RASSF6 were to various degrees methylated in a large portion of neuroblastoma tumors and RASSF7 was heavily methylated in most tumors. Further, CpG methylation sites in the CpG islands of some RASSF family members could be used to significantly discriminate between biological subgroups of neuroblastoma tumors. For example, RASSF5 methylation highly correlated to MYCN amplification and INRG stage M. Furthermore, high methylation of RASSF6 was correlated to unfavorable outcome, 1p deletion and MYCN amplification in our tumor material.

In conclusion
This study shows that several genes belonging to the RASSF gene family are methylated in neuroblastoma. The genes RASSF5, RASSF6 and RASSF7 stand out as the most promising candidate genes for further investigations in neuroblastoma.

Download full-text


Available from: Helena Carén, Feb 14, 2014
  • Source
    • "It has been reported that the global patterns of DNA methylation are altered in many cancers, including HCC [34-36]. To explore the mechanism by HBx and survivin up-regulate HBXIP, we observed the promoter region of HBXIP using epigenetics analysis. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Hepatitis B virus X protein (HBx) plays crucial roles in hepatocarcinogenesis. However, the underlying mechanism remains elusive. We have reported that HBx is able to up-regulate survivin in hepatocellular carcinoma tissues. The oncopreotein hepatitis B X-interacting protein (HBXIP), a target of miR-520b, is involved in the development of cancer. In this study, we focus on the investigation of hepatocarcinogenesis mediated by HBx. Methods The expression of HBx and survivin was examined in the liver tissues of HBx-Tg mice. The effect of HBx/survivin on the growth of LO2-X-S cells was determined by colony formation and transplantation in nude mice. The effect of HBx/survivin on promoter of miR-520b was determined by Western blot analysis, luciferase reporter gene assay, co-immunoprecipitation (co-IP) and chromatin immunoprecipitation (ChIP), respectively. The expression of HBx, survivin and HBXIP was detected by immunohistochemistry and real-time PCR in clinical HCC tissues, respectively. The DNA demethylation of HBXIP promoter was examined. The functional influence of miR-520b and HBXIP on proliferation of hepatoma cells was analyzed by MTT, colony formation, EdU and transplantation in nude mice in vitro and in vivo. Results In this study, we provided evidence that HBx up-regulated survivin in the liver cancer tissues of HBx-Tg mice aged 18 M. The engineered LO2 cell lines with survivin and/or HBx were successfully established, termed LO2-X-S. MiR-520b was down-regulated in LO2-X-S cells and clinical HCC tissues. Our data revealed that HBx survivin-dependently down-regulated miR-520b through interacting with Sp1 in the cells. HBXIP was highly expressed in LO2-X-S cells, liver cancer tissues of HBx-Tg mice aged 18 M and clinical HCC tissues (75.17%, 112/149). The expression level of HBXIP was positively associated with those of HBx or survivin in clinical HCC tissues. In addition, we showed that HBx survivin-dependently up-regulated HBXIP through inducing demethylation of HBXIP promoter in LO2-X-S cells and clinical HCC tissues. In function, low level miR-520b and high level HBXIP mediated by HBx with partner survivin contributed to the growth of LO2-X-S cells in vitro and in vivo. Conclusion HBx accelerates hepatocarcinogenesis with partner survivin through modulating tumor suppressor miR-520b and oncoprotein HBXIP.
    Molecular Cancer 05/2014; 13(1):128. DOI:10.1186/1476-4598-13-128 · 4.26 Impact Factor
  • Source
    • "Rassf4A Widely expressed in most normal tissues (heart, brain, placenta, lung, liver, skeletal muscle, pancreas) Epigenetically silenced in nasopharyngeal carcinoma, breast, lung, colorectal, kidney tumour cell lines Chow et al. (2004); Eckfeld et al. (2004) RASSF5 1q32.1 Rassf5A Rassf5C Widely expressed in normal tissues Rassf5A epigenetically silenced in lung NSCLC, breast, kidney, primary tumours, colorectal, hepatocellular carcinoma, leukaemia, melanoma, neuroblastoma cell lines Calvisi et al. (2009); Lee et al. (2010); Macheiner et al. (2006); van der Weyden and Adams (2007); Djos et al. (2012) RASSF6 4q13.3 Rassf6A Varying levels of expression in colon, thymus, kidney, small intestine, placenta, lung Downregulated by gene deletion and epigenetic silencing in 30–60% of breast, colon, kidney, liver, rectum, pancreas, stomach and thyroid primary tumours Epigenetically silenced in many childhood leukaemias, neuroblastoma Allen et al. (2007); Djos et al. (2012); Hesson et al. (2009) RASSF7 11p15.5 Rassf7A Varying levels of expression in normal tissues, high levels in lung and brain Epigenetically silenced in neuroblastoma Upregulated in pancreatic ductal adecnocarcinoma, islet cell tumours, endometrial cancers due to hypoxia Djos et al. (2012); Recino et al. (2010); Sherwood et al. (2010) "
    [Show abstract] [Hide abstract]
    ABSTRACT: Not all proteins implicated in direct binding to Ras appear to have a positive role in the generation and progression of tumours; examples include Phospholipase C epsilon (PLCɛ) and some members of the Ras-association domain family (RASSF). The RASSF family comprises of ten members, known as RASSF1 to RASSF10. PLCɛ and RASSF members carry a common Ras-association domain (RA) that can potentially bind Ras oncoproteins and mediate Ras-regulated functions. RASSF1 to RASSF6 also share a common SARAH domain that facilitates protein-protein interactions with other SARAH domain proteins. The majority of the family are frequently downregulated by epigenetic silencing in cancers. They are implicated in various important biological processes including apoptosis, microtubule stabilisation and cell cycle regulation. Recent studies have reinforced the tumour suppressive properties of the RASSF family, with new evidence of emerging pathways and novel functions that suggest a wider role for these proteins. This review will first describe an emerging role of PLCɛ in tumour suppression and then focus on and summarise the new findings on the RASSF family in the last five years to consolidate their well-established functions, and highlight the new regulatory roles of specific RASSF members.
    08/2013; 53(3). DOI:10.1016/j.jbior.2013.07.008
  • Source
    • "Promoter hypermethylation induced silencing of RASSF1A, the fist member of the RASSF family, is an early and widespread event in many tumors [13], [16]–[22]. Other members of the RASSF family, such as RASSF2A, RASSF5, RASSF6, RASSF7, and RASSF10, also show frequent DNA methylation in some tumors [23]–[25]. RASSF3 is considered to be responsible in part for resistance to mammary tumor development in neu transgenic mice [26]. However, to the best of our knowledge, there have been no previous studies about the methylation status of RASSF3 in any tumor. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The pathogenic mechanisms underlying pituitary somatotroph adenoma formation, progression are poorly understood. To identify candidate tumor suppressor genes involved in pituitary somatotroph adenoma tumorigenesis, we used HG18 CpG plus Promoter Microarray in 27 human somatotroph adenomas and 4 normal human adenohypophyses. RASSF3 was found with frequent methylation of CpG island in its promoter region in somatotroph adenomas but rarely in adenohypophyses. This result was confirmed by pyrosequencing analysis. We also found that RASSF3 mRNA level correlated negatively to its gene promoter methylation level. RASSF3 hypermethylation and downregulation was also observed in rat GH3 and mouse GT1.1 somatotroph adenoma cell lines. 5-Aza-2' deoxycytidine and trichostatin-A treatment induced RASSF3 promoter demethylation, and restored its expression in GH3 and GT1.1 cell lines. RASSF3 overexpression in GH3 and GT1.1 cells inhibited proliferation, induced apoptosis accompanied by increased Bax, p53, and caspase-3 protein and decreased Bcl-2 protein expression. We also found that the antitumor effect of RASSF3 was p53 dependent, and p53 knockdown blocked RASSF3-induced apoptosis and growth inhibition. Taken together, our results suggest that hypermethylation-induced RASSF3 silencing plays an important role in the tumorigenesis of pituitary somatotroph adenomas.
    PLoS ONE 03/2013; 8(3):e59024. DOI:10.1371/journal.pone.0059024 · 3.23 Impact Factor
Show more