Epigenetic inactivation of the NORE1 gene correlates with malignant progression of colorectal tumors

Chang K. Lee, Jin Hee Lee, Min Goo Lee, Seong In Jeong, Tae Kyu Ha, Min Ju Kang, Byung Kyu Ryu, Young Hwangbo, Jae Jun Shim, Jae Y. Jang, Kil Y. Lee, Hyo J. Kim, Sung-Gil Chi

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Abstract

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.Conclusion: 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.

Original languageEnglish
Article number577
JournalBMC Cancer
Volume10
DOIs
Publication statusPublished - 2010 Oct 22

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Gene Silencing
Epigenomics
Colorectal Neoplasms
Neoplasms
DNA Sequence Analysis
Mutation
Carcinogenesis
Single-Stranded Conformational Polymorphism
Cell Line
Growth
Genetic Promoter Regions
Methylation
Small Interfering RNA
Flow Cytometry
Epithelium
Cell Count
Carcinoma
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Oncology
  • Cancer Research
  • Genetics

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Epigenetic inactivation of the NORE1 gene correlates with malignant progression of colorectal tumors. / Lee, Chang K.; Lee, Jin Hee; Lee, Min Goo; Jeong, Seong In; Ha, Tae Kyu; Kang, Min Ju; Ryu, Byung Kyu; Hwangbo, Young; Shim, Jae Jun; Jang, Jae Y.; Lee, Kil Y.; Kim, Hyo J.; Chi, Sung-Gil.

In: BMC Cancer, Vol. 10, 577, 22.10.2010.

Research output: Contribution to journalArticle

Lee, CK, Lee, JH, Lee, MG, Jeong, SI, Ha, TK, Kang, MJ, Ryu, BK, Hwangbo, Y, Shim, JJ, Jang, JY, Lee, KY, Kim, HJ & Chi, S-G 2010, 'Epigenetic inactivation of the NORE1 gene correlates with malignant progression of colorectal tumors', BMC Cancer, vol. 10, 577. https://doi.org/10.1186/1471-2407-10-577
Lee, Chang K. ; Lee, Jin Hee ; Lee, Min Goo ; Jeong, Seong In ; Ha, Tae Kyu ; Kang, Min Ju ; Ryu, Byung Kyu ; Hwangbo, Young ; Shim, Jae Jun ; Jang, Jae Y. ; Lee, Kil Y. ; Kim, Hyo J. ; Chi, Sung-Gil. / Epigenetic inactivation of the NORE1 gene correlates with malignant progression of colorectal tumors. In: BMC Cancer. 2010 ; Vol. 10.
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abstract = "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.Conclusion: 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.",
author = "Lee, {Chang K.} and Lee, {Jin Hee} and Lee, {Min Goo} and Jeong, {Seong In} and Ha, {Tae Kyu} and Kang, {Min Ju} and Ryu, {Byung Kyu} and Young Hwangbo and Shim, {Jae Jun} and Jang, {Jae Y.} and Lee, {Kil Y.} and Kim, {Hyo J.} and Sung-Gil Chi",
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AU - Lee, Jin Hee

AU - Lee, Min Goo

AU - Jeong, Seong In

AU - Ha, Tae Kyu

AU - Kang, Min Ju

AU - Ryu, Byung Kyu

AU - Hwangbo, Young

AU - Shim, Jae Jun

AU - Jang, Jae Y.

AU - Lee, Kil Y.

AU - Kim, Hyo J.

AU - Chi, Sung-Gil

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N2 - 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.Conclusion: 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.

AB - 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.Conclusion: 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.

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