Abstract
BackgroundNORE1 (RASSF5) is a newly described member of the RASSF family with Ras effector function. NORE1 expression is frequently inactivated by aberrant promoter hypermethylation in many human cancers, suggesting that NORE1 might be a putative tumor suppressor. However, expression and mutation status of NORE1 and its implication in colorectal tumorigenesis has not been evaluated.MethodsExpression, mutation, and methylation status of NORE1A and NORE1B in 10 cancer cell lines and 80 primary tumors were characterized by quantitative PCR, SSCP, and bisulfite DNA sequencing analyses. Effect of NORE1A and NORE1B expression on tumor cell growth was evaluated using cell number counting, flow cytometry, and colony formation assays.ResultsExpression of NORE1A and NORE1B transcript was easily detectable in all normal colonic epithelial tissues, but substantially decreased in 7 (70%) and 4 (40%) of 10 cancer cell lines and 31 (38.8%) and 25 (31.3%) of 80 primary carcinoma tissues, respectively. Moreover, 46 (57.6%) and 38 (47.5%) of 80 matched tissue sets exhibited tumor-specific reduction of NORE1A and NORE1B, respectively. Abnormal reduction of NORE1 was more commonly observed in advanced stage and high grade tumors compared to early and low grade tumors. While somatic mutations of the gene were not identified, its expression was re-activated in all low expressor cells after treatment with the demethylating agent 5-aza-dC. Bisulfite DNA sequencing analysis of 31 CpG sites within the promoter region demonstrated that abnormal reduction of NORE1A is tightly associated with promoter CpG sites hypermethylation. Moreover, transient expression and siRNA-mediated knockdown assays revealed that both NORE1A and NORE1B decrease cellular growth and colony forming ability of tumor cells and enhance tumor cell response to apoptotic stress.ConclusionOur data indicate that epigenetic inactivation of NORE1 due to aberrant promoter hypermethylation is a frequent event in colorectal tumorigenesis and might be implicated in the malignant progression of colorectal tumors.
Highlights
NORE1 (RASSF5) is a newly described member of the Rasassociation domain family (RASSF) family with Ras effector function
Frequent reduction of NORE1 expression in primary carcinoma tissues and cancer cell lines To explore whether NORE1 alteration is implicated in colorectal tumorigenesis, we initially determined expression levels of NORE1 transcripts in 80 noncancerous tissues and 10 cancer cell lines using semi-quantitative reverse transcription (RT)-PCR analysis
Expression of NORE1A and NORE1B transcripts was detectable in all noncancerous tissues tested and its levels showed no significant variations among the specimens (Figure 1A)
Summary
NORE1 (RASSF5) is a newly described member of the RASSF family with Ras effector function. NORE1 expression is frequently inactivated by aberrant promoter hypermethylation in many human cancers, suggesting that NORE1 might be a putative tumor suppressor. Among 8 RASSF genes identified, RASSF1 was the most well-characterized tumor suppressor, which exhibits a frequent epigenetic inactivation in various types of human neoplasms, including lung and breast cancers [6,7,8,9,10,11]. Transcriptional silencing of RASSF1A was observed in a considerable proportion of lung, breast, ovarian, and nasopharyngeal cancers by de novo methylation at the CpG islands in the promoter [8,9,10,11]. In small cell lung cancers, allelic loss of 3p21.3 was associated with RASSF1A methylation, suggesting that both genetic and epigenetic mechanisms are implicated in RASSF1A inactivation in some tumor types [9]. RASSF1A was identified to suppress the growth of tumor cells in both in vitro and in vivo, and Ras regulates pro-apoptotic pathway through its interaction with RASSF1A [5,8,12]
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