Epigenetic inactivation of the RASSF10 candidate tumor suppressor gene is a frequent and an early event in gliomagenesis

Abstract

We have recently described the N-terminal RAS association domain family of genes, RASSF7-10. Previously, we cloned the N-terminal RASSF10 gene and demonstrated frequent methylation of the associated 5 0 -CpG island in acute lymphoblastic leukemia. To characterize RASSF10 gene expression, we demonstrate that in developing Xenopus embryos, RASSF10 shows a very striking pattern in the rhombencephalon (hind brain). It is also expressed in other parts of the brain and other organs. Due to the well-defined expression pattern in the brain of Xenopus embryos, we analyzed the methylation status of the RASSF10-associated 5 0 -CpG island in astrocytic gliomas. RASSF10 was frequently methylated in WHO grade II-III astrocytomas and WHO grade IV primary glioblastomas (67.5%), but was unmethylated in grade I astrocytomas and in DNA from age matched control brain samples. RASSF10 gene expression both at the mRNA and protein levels could be switched back on in methylated glioma cell lines after treatment with 5-aza-2 0 -deoxycytidine. In secondary glioblastomas (sGBM), RASSF10 methylation was an independent prognostic factor associated with worst progression-free survival and overall survival and occurred at an early stage in their development. In cell culture experiments, overexpression of RASSF10 mediated a reduction in the colony forming ability of two RASSF10-methylated glioma cell lines. Conversely, RNAi-mediated knockdown of RASSF10-stimulated anchorage-independent growth of U87 glioma cells, increased their viability and caused an increase in the cells' proliferative ability. We generated and characterized a RASSF10-specific antibody and demonstrated for the first time that RASSF10 subcellular localization is cell-cycle dependent with RASSF10 colocalizing to centrosomes and associated microtubules during mitosis. This is the first report demonstrating that RASSF10 can act as a tumor suppressor gene and is frequently methylated in gliomas and can potentially be developed into a prognostic marker for sGBM.

RASSF tumor suppressor gene family: Biological functions and regulation

“…RASSF10 was shown to be expressed in bone marrow as well as thyroid, brain, prostate and kidney [91,[113][114][115]. Concomitantly, hypermethylation of RASSF10 was associated with loss of gene and protein expression in cancer conditions such as childhood leukaemias, thyroid carcinomas and gliomas [91,113,114]. The transcription of RASSF10 was restored using a demethylating agent [116].…”

“…The pro-apoptotic nature of RASSF10 was shown to involve the Wnt/b-catenin signaling pathway [116]. Additionally, RASSF10 may play a potential role in the regulation of mitotic progression due to its localization to centrosomes and microtubule association [113]. In light of the existing information, it appears that RASSF10 is a strong candidate as a prognostic marker to screen certain carcinomas.…”

“…The transcription of RASSF10 was restored using a demethylating agent [116]. In vitro analyses in U87 glioma cells treated with RNAi to knockdown RASSF10 expression, showed increased cell proliferation and cell survival [113]. Re-expression of RASSF10 in these cells inhibited cell growth and colony formation of glioma cells highlighting the tumor suppressor function of RASSF10 [113].…”

“…In vitro analyses in U87 glioma cells treated with RNAi to knockdown RASSF10 expression, showed increased cell proliferation and cell survival [113]. Re-expression of RASSF10 in these cells inhibited cell growth and colony formation of glioma cells highlighting the tumor suppressor function of RASSF10 [113]. More recently, the tumor suppressor activity of RASSF10 was assessed in gastric cancer cells, demonstrating that reintroduction of RASSF10 in gastric cell lines JRST and BGC823 reduced cell viability and promoted apoptosis [116].…”

Epigenetic mechanisms regulating neural development and pediatric brain tumor formation

“…121 In another study, the N-terminal RAS association domain family gene (RASSF7-10) was described as a tumor suppressor gene and was found to be frequently methylated in gliomas, and was associated with a poor prognosis. 61 In addition, cancer cells also display widespread changes in histone markers: deacetylation of histones H3 and H4 is mediated by HDACs and results in gene repression; gain of H3K9 methylation and H3K27 trimethylation is mediated by HMTs and leads to aberrant silencing of tumor suppressor genes. 129 One particularly important example of epigenetic silencing is the promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma.…”