Frequent epigenetic inactivation of RASSF1A and BLU genes located within the critical 3p21.3 region in gliomas

Abstract

RASSF1A is a major tumor suppressor gene located at 3p21.3. We investigated the role of aberrant promoter region hypermethylation of RASSF1A in a large series of adult gliomas. RASSF1A was frequently methylated in both primary tumors (36/63; 57%) and tumor cell lines (7/7; 100%). Hypermethylation of RASSF1A in glioma cell lines correlated with loss of expression and treatment with a demethylating agentreactivated RASSF1A gene expression. Furthermore, reexpression of RASSF1A suppressed the growth of glioma cell line H4 in vitro. Next, we investigated whether other members of the RASSF gene family were also inactivated by methylation. NORE1B and RASSF3 were not methylated in gliomas, while NORE1A and RASSF5/ AD037 demonstrated methylation in glioma cell lines but not in primary tumors. We then investigated the methylation status of three other candidate 3p21.3 tumor suppressor genes. CACNA2D2 and SEMA3B were not frequently methylated, but the BLU gene located just centromeric to RASSF1 was frequently methylated in glioma cell lines (7/7) and in 80% (35/44) of glioma tumors. In these tumor cell lines, BLU expression was restored after treatment with a demethylating agent. Loss of BLU gene expression in glioma tumors correlated with BLU methylation. There was no association between RASSF1A and BLU methylation. RASSF1A methylation increased with tumor grade, while BLU methylation was seen at similar frequencies in all grades. Our data implicate RASSF1A and BLU promoter methylation in the pathogenesis of adult gliomas, while other RASSF family members and CACNA2D2 and SEMA3B appear to have only minor roles. In addition, RASSF1A and BLU methylation appear to be independent and specific events and not due to region-wide changes in DNA methylation.

Identification of a tumor suppressive critical region mapping to 3p14.2 in esophageal squamous cell carcinoma and studies of a candidate tumor suppressor gene, ADAMTS9

“…Numerous studies in lung (Sekine et al, 2005), hepatocellular (Tischoff et al, 2005), NPC (Xiong et al, 2004), glioma (Hesson et al, 2004), and brain tumors (Horiguchi et al, 2003) suggest that chromosome 3 harbors TSGs. Positional cloning and molecular and cytogenetic approaches identified possible tumor suppressive regions mapping to 3p23 (Wang et al, 1996), 3p14.2 and 3p21.3 (Ko et al, 2001), and 3p21.1-p21.2 (Shiomi et al, 2003) in ESCC.…”

Evaluation of the 3p21.3 tumour-suppressor gene cluster

“…Mutation of BLU/ZMYND10 is a rare event in lung cancer since missense changes were detected in only 3/61 lung tumour-cell lines (Lerman and Minna, 2000). Interestingly, however, expression of BLU/ZMYND10 is lost or downregulated in a subset of NPC, lung, neuroblastoma, kidney, breast, esophageal squamous cell carcinoma and glioma tumour-cell lines (Lerman and Minna, 2000;Agathanggelou et al, 2003b;Hesson et al, 2004;Qiu et al, 2004;Yau et al, 2006;Yi Lo et al, 2006). Promoter CpG island hypermethylation is coincident with loss of BLU/ZMYND10 expression, which can be restored following treatment with the DNA demethylating agent 5-aza-2 0 -deoxycytidine (5azaDC) (Agathanggelou et al, 2003b;Hesson et al, 2004;Qiu et al, 2004;Yau et al, 2006;Yi Lo et al, 2006).…”

“…Interestingly, however, expression of BLU/ZMYND10 is lost or downregulated in a subset of NPC, lung, neuroblastoma, kidney, breast, esophageal squamous cell carcinoma and glioma tumour-cell lines (Lerman and Minna, 2000;Agathanggelou et al, 2003b;Hesson et al, 2004;Qiu et al, 2004;Yau et al, 2006;Yi Lo et al, 2006). Promoter CpG island hypermethylation is coincident with loss of BLU/ZMYND10 expression, which can be restored following treatment with the DNA demethylating agent 5-aza-2 0 -deoxycytidine (5azaDC) (Agathanggelou et al, 2003b;Hesson et al, 2004;Qiu et al, 2004;Yau et al, 2006;Yi Lo et al, 2006). BLU/ZMYND10 hypermethylation and downregulation has been described as a frequent event in primary tumours such as glioma (80%), cervical squamous cell carcinomas (77%), NPC (66%), neuroblastoma (41-70%) and NSCLC (19-43%) with lower frequencies observed in gall bladder carcinomas (26%), ependymomas (13.6%) and SCLC (14%) (Agathanggelou et al, 2003b;Hesson et al, 2004;Qiu et al, 2004;Abe et al, 2005;Ito et al, 2005;Marsit et al, 2005;Lai et al, 2006;Michalowski et al, 2006;Riquelme et al, 2007).…”

“…Promoter CpG island hypermethylation is coincident with loss of BLU/ZMYND10 expression, which can be restored following treatment with the DNA demethylating agent 5-aza-2 0 -deoxycytidine (5azaDC) (Agathanggelou et al, 2003b;Hesson et al, 2004;Qiu et al, 2004;Yau et al, 2006;Yi Lo et al, 2006). BLU/ZMYND10 hypermethylation and downregulation has been described as a frequent event in primary tumours such as glioma (80%), cervical squamous cell carcinomas (77%), NPC (66%), neuroblastoma (41-70%) and NSCLC (19-43%) with lower frequencies observed in gall bladder carcinomas (26%), ependymomas (13.6%) and SCLC (14%) (Agathanggelou et al, 2003b;Hesson et al, 2004;Qiu et al, 2004;Abe et al, 2005;Ito et al, 2005;Marsit et al, 2005;Lai et al, 2006;Michalowski et al, 2006;Riquelme et al, 2007). In some tumour types, BLU/ZMYND10 methylation is an early event detectable in stage II glioma and stage I/II cervical squamous cell carcinomas (at frequencies of 88 and 85/68%, respectively) Lai et al, 2006).…”

Induction of tumor inhibition and apoptosis by a candidate tumor suppressor gene DRR1 on 3p21.1

“…Alterations of human 3p frequently occur in many types of cancer, including lung carcinoma. Several studies have proven tumor-acquired promoter hypermethylation as a mechanism of inactivation of mRNA expression of some TSGs on 3p during pathogenesis of human lung cancer and several other cancers (5,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). Given the fact that no genetic alterations were identified in DRR1, it was suggested that epigenetic alteration such as promoter methylation may account for the inactivity of allelic loss (3,4).…”