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

Molecular Cancer (Impact Factor: 5.4). 06/2012; 11(1):40. DOI: 10.1186/1476-4598-11-40
Source: PubMed

ABSTRACT Background
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
    • "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; DOI:10.1016/j.jbior.2013.07.008
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ras association (RalGDS/AF-6) domain family member RASSF5 is a non-enzymatic RAS effector super family protein, known to be involved in cell growth regulation. Expression of RASSF5 is found to be extinguished by promoter hypermethylation in different human cancers, and its ectopic expression suppresses cell proliferation and tumorigenicity. Interestingly, this role in tumorigenesis has been confounded by the fact that regulation at molecular level remains unclear and many transformed cells actually display elevated RASSF5 expression. Here, we demonstrate that E3 ubiquitin ligase Itch is a unique binding partner of RASSF5. Itch can interact with PPxY motif in RASSF5 both in vivo and in vitro through its WW domains. Importantly, the overexpression of Itch induces RASSF5 degradation by poly-ubiquitination via 26S proteasome pathway. In addition, our results indicate that the elevated levels of RASSF5 found in tumor cells due to acetylation, which restricts its binding to Itch and results in a more stable inert protein. Inhibition of RASSF5 acetylation permits its interaction with Itch and provokes proteasomal degradation. These data suggest that apart from promoter methylation, hyperacetylation could also be downregulating RASSF5 function in different human cancer. Finally, results from functional assays suggest that the overexpression of wild type, not the ligase activity defective Itch negatively regulate RASSF5-mediated G1 phase transition of cell cycle as well as apoptosis, suggesting that Itch alone is sufficient to alter RASSF5 function. Collectively, the present investigation identifies a HECT class E3 ubiquitin ligase Itch as a unique negative regulator of RASSF5, and suggests the possibility that acetylation as a potential therapeutic target for human cancer.
    Cell Death & Disease 03/2013; 4(3):e565. DOI:10.1038/cddis.2013.91 · 5.18 Impact Factor
  • Source
    [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 · 3.53 Impact Factor
Show more