Epigenetic Regulation Identifies RASEF as a Tumor-Suppressor Gene in Uveal Melanoma (original) (raw)
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Epigenetic Inactivation of RASSF1a in Uveal Melanoma
Investigative Ophthalmology & Visual Science, 2007
PURPOSE. The RAS association domain family 1 (RASSF1) gene is a tumor-suppressor gene located on chromosome 3p21.3. The alternative transcript (RASSF1a) has been shown to be inactivated by hypermethylation in several human malignancies, including breast, prostate, and lung cancer, and in cutaneous melanoma. The purpose of this study was to evaluate the methylation status of RASSF1a in human uveal melanoma. METHODS. The methylation status of the RASSF1a promoter region was analyzed using PCR in combination with melting curve analysis, sequencing, and restriction enzyme analysis. Eleven human uveal melanoma cell lines, normal melanocytes, 39 archival frozen tumor specimens, and a metastatic lesion of untreated primary uveal melanoma were studied. In addition, whether RASSF1a methylation correlates with patient survival and development of metastatic disease was investigated. RESULTS. RASSF1a promoter methylation was detected in 10 of the 11 (91%) cell lines, in 19 of the 38 (50%) patients with primary uveal melanoma and in the metastatic lesion. A positive correlation was found between RASSF1a promoter methylation and development of metastatic disease (P ϭ 0.041). A correlation with disease-free survival could not be established, but a positive trend was observed (P ϭ 0.063). CONCLUSIONS. These data show that RASSF1a methylation is a common epigenetic event in uveal melanoma development, potentially of clinical relevance. The presence of a methylated RASSF1a promoter region might therefore serve as a tumor marker and as a possible target for therapeutic intervention. (Invest Ophthalmol Vis Sci. 2007;48: 486 -490)
Promoter Methylation Status of Multiple Genes in Uveal Melanoma
Investigative Ophthalmology & Visual Science, 2007
PURPOSE. Aberrant promoter hypermethylation of CpG islands is thought to play an important role in the inactivation of tumor-suppressor genes (TSGs) in cancer. Studies of cutaneous melanoma have reported a high methylation rate for MGMT, DAPK, RAR-b2, and RASSF1A. In colon cancer, SOCS-1, IGF-2, RUNX3, NEUROG1, and CACNA1G are commonly inactivated. The concomitant methylation of at least three of these genes may represent a distinct trait, the CpG island methylator phenotype (CIMP). The purpose of the present study was to investigate the role of epigenetic inactivation of multiple genes in uveal melanoma. METHODS. Twenty samples of uveal melanoma were analyzed for the methylation status of nine candidate cancer-related genes: MGMT, DAPK, RAR-b2, RASSF1A, SOCS-1, IGF-2, RUNX3, NEUROG1, and CACNA1G, using real-time quantitative methylation-specific polymerase chain reaction after sodium bisulfite modification. RESULTS. Methylation rates of the genes commonly inactivated in cutaneous melanoma were 70% for RASSFIA, 5% for MGMT and DAPK, and 0 for RAR-b2. The rates for the CIMP-related genes were 25% for RUNX3, 5% for NEUROG1 and CACNA1G, and 0 for SOCS-1 and IGF-2. None of the samples was CIMPpositive. CONCLUSIONS. In this study uveal melanoma was negative for CIMP, with hypermethylation of RASSF1A. The negative CIMP phenotype and frequent RASSF1A methylation in uveal melanoma is in accord with its known lack of BRAF mutations. Given that mutations in genes of the RAS pathway are rarely observed in uveal melanoma, epigenetic inactivation of RASSF1A may be an alternative mechanism of tumorigenesis.
Methylation of CpG island promoters in uveal melanoma
British Journal of Ophthalmology, 2008
Background: Inactivation of tumour-related genes by promoter hypermethylation is a common epigenetic event in the development of a variety of tumours. Aim: To investigate in primary uveal melanoma the status of promoter methylation of genes thought to be involved in tumour development: p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC. Methods: Gene promoter methylation was studied by methylation-sensitive single-strand conformation analysis and dot-blot assay in a series of 23 primary uveal melanomas. All DNA samples were obtained from paraffin-embedded formalin-fixed tissue blocks. Results: hTERT promoter methylation was found with a relatively high frequency (52%). Promoter methylation of p16, TIMP3, RASSF1, RARB, FHIT and APC was a rare event. For none of these genes did promoter methylation exceed 15% of tumour samples, and, for some genes (FHIT and APC), no methylation was found at all. Furthermore, promoter methylation was absent in 39% (9/ 23) of cases. In only 22% (5/23) of cases was hypermethylation of at least two promoters observed. Conclusions: Promoter methylation of hTERT is a regular event in uveal melanoma. Hypermethylation of the other genes studied does not seem to be an essential element in the development of this tumour. As promoter methylation of APC, RASSF1 and RARB is often observed in cutaneous melanoma, these results suggest that different epigenetic events occur in the development of cutaneous and uveal melanoma. Figure 2 (A) Methylation analysis of p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC promoters in 23 uveal melanomas. The numbers identify the individual cases. The methylation analysis was performed using methylation-sensitive single-strand conformation analysis (MS-SSCA) and methylation-sensitive dot-blot assay (MS-DBA). (B) Percentage of methylated samples for each gene. , Br J Ophthalmol http://bjo.bmj.com/content/92/2/281
Status of RASSF1A in Uveal Melanocytes and Melanoma Cells
Molecular Cancer Research, 2011
RASSF1A gene, found at the 3p21.3 locus, is a tumor suppressor gene frequently hypermethylated in human cancers. In this study, we report that compared with melanocytes in normal choroid, RASSF1A is downregulated in uveal melanoma samples and in uveal melanoma cell lines. LOH at 3p21.3 was detected in 50% of uveal melanoma. Moreover, methylation of the RASSF1A promoter was detected in 35 of 42 tumors (83%) and RASSF1A was also weakly expressed at the mRNA level. These data indicate that LOH at the RASSF1A locus or RASSF1A promoter methylation may partly account for the suppression of RASSF1A expression observed in uveal melanoma. Furthermore, following ectopic expression in three RASSF1A-deficient melanoma cell lines (OCM-1, Mel270, and 92.1), RASSF1A weakly reduces cell proliferation and anchorage-independent growth of uveal melanoma cells without effect on ERK1/2 activation, cyclin D1 and p27 Kip1 expression. This study explored biological functions and underlying mechanisms of RASSF1A in the ERK1/2 pathway in normal uveal melanocytes. We showed that siRNAmediated depletion of RASSF1A increased ERK1/2 activation, cyclin D1 expression, and also decreased p27 Kip1 expression in normal uveal melanocytes. Moreover, that the depletion of RASSF1A induced senescence-associated b-galactosidase activity and increased p21 Cip1 expression suggests that RASSF1A plays a role in the escape of cellular senescence in normal uveal melanocytes. Interestingly, we found that RASSF1A was epigenetically inactivated in long-term culture of uveal melanocytes. Taken together, these data show that depletion of RASSF1A could be an early event observed during senescence of normal uveal melanocytes and that additional alterations are acquired during malignant transformation to uveal melanoma. Mol Cancer Res; 9(9);
The Role of RASSF1A in Uveal Melanoma
Investigative Ophthalmology & Visual Science, 2012
PURPOSE. RASSF1A inactivation in uveal melanoma (UM) is common and methylation-induced. This study investigated the effect of RASSF1A reexpression on the UM phenotype in vivo and in vitro.
Aberrant DNA methylation of uveal melanoma drivers as an excellent prognostic tool
Research Square (Research Square), 2023
Background: Despite outstanding advances in understanding the genetic background of uveal melanoma (UM) development and prognosis, the role of DNA methylation reprogramming remains elusive. This study aims to clarify the extent of DNA methylation deregulation in the context of gene expression changes and its utility as a reliable prognostic biomarker. Methods: Transcriptomic and DNA methylation landscapes in 25 high-and low-risk UMs were interrogated by Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarray and Human In nium Methylation EPIC Bead Chip array, respectively. DNA methylation and gene expression of the nine top discriminatory genes, selected by the integrative analysis, were validated by pyrosequencing and qPCR in 58 tissues. Results: Among 2,262 differentially expressed genes discovered in UM samples differing in metastatic risk, 60 were epigenetic regulators, mostly histone modi ers and chromatin remodelers. A total of 44,398 CpGs were differentially methylated, 27,810 hypomethylated, and 16,588 hypermethylated in high-risk tumors, with Δβ values ranging between-0.78 and 0.79. By integrative analysis, 944 differentially expressed DNA methylation-regulated genes were revealed, 635 hypomethylated/upregulated, and 309 hypermethylated/downregulated. Aberrant DNA methylation in high-risk tumors was associated with the deregulation of key oncogenic pathways such as EGFR tyrosine kinase inhibitor resistance, focal adhesion, proteoglycans in cancer, PI3K-Akt signaling, or ECM-receptor interaction. Notably, the DNA methylation values of nine genes, HTR2B, AHNAK2, CALHM2, SLC25A38, EDNRB, TLR1, RNF43, IL12RB2, and MEGF10, validated by pyrosequencing, demonstrated excellent risk group prediction accuracies (AUCs ranging between 0.870 and 0.956). Moreover, CALHM2 hypomethylation and MEGF10, TLR1 hypermethylation, as well as two three-gene methylation signatures, Signature 1 combining AHNAK2, CALHM2, and IL12RB and Signature 2 AHNAK2, CALHM2, and SLC25A38 genes, correlated with shorter overall survival (
DNA methylation aberrancy is a reliable prognostic tool in uveal melanoma
Background: Despite outstanding advances in understanding the genetic background of uveal melanoma (UM) development and prognosis, the role of DNA methylation reprogramming remains elusive. This study aims to clarify the extent of DNA methylation deregulation in the context of gene expression changes and its utility as a reliable prognostic biomarker. Methods: Transcriptomic and DNA methylation landscapes in 25 high- and low-risk UMs were interrogated by Agilent SurePrint G3 Human Gene Expression 8×60K v2 Microarray and Human Infinium Methylation EPIC Bead Chip array, respectively. DNA methylation and gene expression of the nine top discriminatory genes, selected by the integrative analysis, were validated by pyrosequencing and qPCR in 58 tissues. Results: Among 2,262 differentially expressed genes discovered in UM samples differing in metastatic risk, 60 were epigenetic regulators, mostly histone modifiers and chromatin remodelers. A total of 44,398 CpGs were differentially methyla...
Oncotarget, 2016
We previously identified PRAME as a biomarker for metastatic risk in Class 1 uveal melanomas. In this study, we sought to define a threshold value for positive PRAME expression (PRAME+) in a large dataset, identify factors associated with PRAME expression, evaluate the prognostic value of PRAME in Class 2 uveal melanomas, and determine whether PRAME expression is associated with aberrant hypomethylation of the PRAME promoter. Among 678 samples analyzed by qPCR, 498 (73.5%) were PRAME- and 180 (26.5%) were PRAME+. Class 1 tumors were more likely to be PRAME-, whereas Class 2 tumors were more likely to be PRAME+ (P < 0.0001). PRAME expression was associated with shorter time to metastasis and melanoma specific mortality in Class 2 tumors (P = 0.01 and P = 0.02, respectively). In Class 1 tumors, PRAME expression was directly associated with SF3B1 mutations (P < 0.0001) and inversely associated with EIF1AX mutations (P = 0.004). PRAME expression was strongly associated with hypome...