Epigenetic inactivation of the NORE1 gene correlates with malignant progression of colorectal tumors.
BACKGROUND
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. However, expression and mutation status of NORE1 and its implication in colorectal tumorigenesis has not been evaluated.


METHODS
Expression, 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.


RESULTS
Expression 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.


CONCLUSIONS
Our 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.
Lee
Chang Kyun
CK
Division of Gastroenterology, Department of Internal Medicine, Kyung Hee University School of Medicine, Seoul, Korea.
Lee
Jin-Hee
JH
Lee
Min-Goo
MG
Jeong
Seong-In
SI
Ha
Tae-Kyu
TK
Kang
Min-Ju
MJ
Ryu
Byung-Kyu
BK
Hwangbo
Young
Y
Shim
Jae-Jun
JJ
Jang
Jae Young
JY
Lee
Kil Yeon
KY
Kim
Hyo Jong
HJ
Chi
Sung-Gil
SG
D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D018450:Disease Progression; D044127:Epigenesis, Genetic; D005434:Flow Cytometry / Q000379:methods; D016147:Genes, Tumor Suppressor; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C454042:RASSF5 protein, human; D020559:Monomeric GTP-Binding Proteins