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Promoter DNA methylation of oncostatin m receptor-beta as a novel diagnostic and therapeutic marker in colon cancer.

Kim MS, Louwagie J, Carvalho B, Terhaar Sive Droste JS, Park HL, Chae YK, Yamashita K, Liu J, Ostrow KL, Ling S, Guerrero-Preston R, Demokan S, Yalniz Z, Dalay N, Meijer GA, Van Criekinge W, Sidransky D - PLoS ONE (2009)

Bottom Line: We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100).Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease.Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging "cancer methylome". In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-beta (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

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Promoter methylation analysis.A, Methylation status of each gene was examined in CRC cell lines and tissues by C-MSP after PCR with methylation-specific primers. PCR products were run on 4% agarose gels pre-stained with ethidium bromide. In vitro methylated, bisulfite-treated human normal lymphocyte DNA (NL) was used as a positive control for PCR, and distilled water (DW) was used as a negative PCR control. β-actin was used to confirm integrity of DNA. SLC39A4 and SLC9A3R1 were methylated both in cell lines and tissues, but GANAB, PLAU and UBE3A were not. The other 5 genes were methylated in one of three cells lines tested, and only methylated in CRC tissues (PT). PN, paired normal colon tissues from colon cancer patients; PT, paired CRC; NN, normal colon epithelium from non-cancer patients. C-MSP results of OSMR in CRC cell lines and tissues were shown in a previous report [12]. B, Scatter plots of methylation values of six candidate genes in tissues and cell lines (CL). The overall methylation values (TaqMan methylation values, TaqMeth V) and the optimal specificity/sensitivity at optimal cut-offs calculated from ROC analysis are shown in Table 3. Arrows indicate the optimal cut-off values for each gene. No cases of NN displayed TaqMeth V over the optimal cut-offs for B4GALT1 and OSMR. HEK293, a non-tumorigenic cell line, harbored a high level of B4GALT1 methylation (TaqMeth V, 101.12), but OSMR methylation was not detected in the cell line (TaqMeth V, 0.00). *, Samples with a ratio equal to zero could not be plotted correctly on a log scale, so are presented here as 0.1. All assays were performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results in triplicate. TaqMeth V is described in Materials and Methods. C, Quantitative methylation levels of B4GALT1 and OSMR in primary colon tissues. Scatter plot of B4GALT1 and OSMR promoter methylation. Arrows indicate optimal cut-off values for each gene (3.877 for B4GALT1 and 22.01 for OSMR).
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pone-0006555-g001: Promoter methylation analysis.A, Methylation status of each gene was examined in CRC cell lines and tissues by C-MSP after PCR with methylation-specific primers. PCR products were run on 4% agarose gels pre-stained with ethidium bromide. In vitro methylated, bisulfite-treated human normal lymphocyte DNA (NL) was used as a positive control for PCR, and distilled water (DW) was used as a negative PCR control. β-actin was used to confirm integrity of DNA. SLC39A4 and SLC9A3R1 were methylated both in cell lines and tissues, but GANAB, PLAU and UBE3A were not. The other 5 genes were methylated in one of three cells lines tested, and only methylated in CRC tissues (PT). PN, paired normal colon tissues from colon cancer patients; PT, paired CRC; NN, normal colon epithelium from non-cancer patients. C-MSP results of OSMR in CRC cell lines and tissues were shown in a previous report [12]. B, Scatter plots of methylation values of six candidate genes in tissues and cell lines (CL). The overall methylation values (TaqMan methylation values, TaqMeth V) and the optimal specificity/sensitivity at optimal cut-offs calculated from ROC analysis are shown in Table 3. Arrows indicate the optimal cut-off values for each gene. No cases of NN displayed TaqMeth V over the optimal cut-offs for B4GALT1 and OSMR. HEK293, a non-tumorigenic cell line, harbored a high level of B4GALT1 methylation (TaqMeth V, 101.12), but OSMR methylation was not detected in the cell line (TaqMeth V, 0.00). *, Samples with a ratio equal to zero could not be plotted correctly on a log scale, so are presented here as 0.1. All assays were performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results in triplicate. TaqMeth V is described in Materials and Methods. C, Quantitative methylation levels of B4GALT1 and OSMR in primary colon tissues. Scatter plot of B4GALT1 and OSMR promoter methylation. Arrows indicate optimal cut-off values for each gene (3.877 for B4GALT1 and 22.01 for OSMR).

Mentions: To identify genes harboring cancer-specific promoter methylation, we examined the methylation status of the 23 genes in five to ten pairs of matched primary CRC (PT) and corresponding normal colon (PN) tissues. Normal colon mucosa tissues from non-cancer patients (NN) were also included to compare methylation specificity between cancer and non-cancer patients. We excluded genes that harbored methylation in NN with frequencies higher than 30%. As a result, we found that 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2), γ-tubulin gene 2 (TUBG2), NTRK2, B4GALT1, and OSMR as well as SFRP4 harbored cancer-specific methylation with high frequencies (>30%) (Table 2). All 6 of these genes did not harbor methylation in all NN tested, and differences in NN vs. PT and methylation vs. unmethylation cases were statistically significant. Representative results of C-MSP, bisulfite-sequencing, and COBRA in cell lines and tissues are shown in Figure 1A and Figure S2.


Promoter DNA methylation of oncostatin m receptor-beta as a novel diagnostic and therapeutic marker in colon cancer.

Kim MS, Louwagie J, Carvalho B, Terhaar Sive Droste JS, Park HL, Chae YK, Yamashita K, Liu J, Ostrow KL, Ling S, Guerrero-Preston R, Demokan S, Yalniz Z, Dalay N, Meijer GA, Van Criekinge W, Sidransky D - PLoS ONE (2009)

Promoter methylation analysis.A, Methylation status of each gene was examined in CRC cell lines and tissues by C-MSP after PCR with methylation-specific primers. PCR products were run on 4% agarose gels pre-stained with ethidium bromide. In vitro methylated, bisulfite-treated human normal lymphocyte DNA (NL) was used as a positive control for PCR, and distilled water (DW) was used as a negative PCR control. β-actin was used to confirm integrity of DNA. SLC39A4 and SLC9A3R1 were methylated both in cell lines and tissues, but GANAB, PLAU and UBE3A were not. The other 5 genes were methylated in one of three cells lines tested, and only methylated in CRC tissues (PT). PN, paired normal colon tissues from colon cancer patients; PT, paired CRC; NN, normal colon epithelium from non-cancer patients. C-MSP results of OSMR in CRC cell lines and tissues were shown in a previous report [12]. B, Scatter plots of methylation values of six candidate genes in tissues and cell lines (CL). The overall methylation values (TaqMan methylation values, TaqMeth V) and the optimal specificity/sensitivity at optimal cut-offs calculated from ROC analysis are shown in Table 3. Arrows indicate the optimal cut-off values for each gene. No cases of NN displayed TaqMeth V over the optimal cut-offs for B4GALT1 and OSMR. HEK293, a non-tumorigenic cell line, harbored a high level of B4GALT1 methylation (TaqMeth V, 101.12), but OSMR methylation was not detected in the cell line (TaqMeth V, 0.00). *, Samples with a ratio equal to zero could not be plotted correctly on a log scale, so are presented here as 0.1. All assays were performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results in triplicate. TaqMeth V is described in Materials and Methods. C, Quantitative methylation levels of B4GALT1 and OSMR in primary colon tissues. Scatter plot of B4GALT1 and OSMR promoter methylation. Arrows indicate optimal cut-off values for each gene (3.877 for B4GALT1 and 22.01 for OSMR).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2717211&req=5

pone-0006555-g001: Promoter methylation analysis.A, Methylation status of each gene was examined in CRC cell lines and tissues by C-MSP after PCR with methylation-specific primers. PCR products were run on 4% agarose gels pre-stained with ethidium bromide. In vitro methylated, bisulfite-treated human normal lymphocyte DNA (NL) was used as a positive control for PCR, and distilled water (DW) was used as a negative PCR control. β-actin was used to confirm integrity of DNA. SLC39A4 and SLC9A3R1 were methylated both in cell lines and tissues, but GANAB, PLAU and UBE3A were not. The other 5 genes were methylated in one of three cells lines tested, and only methylated in CRC tissues (PT). PN, paired normal colon tissues from colon cancer patients; PT, paired CRC; NN, normal colon epithelium from non-cancer patients. C-MSP results of OSMR in CRC cell lines and tissues were shown in a previous report [12]. B, Scatter plots of methylation values of six candidate genes in tissues and cell lines (CL). The overall methylation values (TaqMan methylation values, TaqMeth V) and the optimal specificity/sensitivity at optimal cut-offs calculated from ROC analysis are shown in Table 3. Arrows indicate the optimal cut-off values for each gene. No cases of NN displayed TaqMeth V over the optimal cut-offs for B4GALT1 and OSMR. HEK293, a non-tumorigenic cell line, harbored a high level of B4GALT1 methylation (TaqMeth V, 101.12), but OSMR methylation was not detected in the cell line (TaqMeth V, 0.00). *, Samples with a ratio equal to zero could not be plotted correctly on a log scale, so are presented here as 0.1. All assays were performed in duplicate format, and experiments were repeated twice. Data showed reproducible and concordant results in triplicate. TaqMeth V is described in Materials and Methods. C, Quantitative methylation levels of B4GALT1 and OSMR in primary colon tissues. Scatter plot of B4GALT1 and OSMR promoter methylation. Arrows indicate optimal cut-off values for each gene (3.877 for B4GALT1 and 22.01 for OSMR).
Mentions: To identify genes harboring cancer-specific promoter methylation, we examined the methylation status of the 23 genes in five to ten pairs of matched primary CRC (PT) and corresponding normal colon (PN) tissues. Normal colon mucosa tissues from non-cancer patients (NN) were also included to compare methylation specificity between cancer and non-cancer patients. We excluded genes that harbored methylation in NN with frequencies higher than 30%. As a result, we found that 3′-phosphoadenosine 5′-phosphosulfate synthase 2 (PAPSS2), γ-tubulin gene 2 (TUBG2), NTRK2, B4GALT1, and OSMR as well as SFRP4 harbored cancer-specific methylation with high frequencies (>30%) (Table 2). All 6 of these genes did not harbor methylation in all NN tested, and differences in NN vs. PT and methylation vs. unmethylation cases were statistically significant. Representative results of C-MSP, bisulfite-sequencing, and COBRA in cell lines and tissues are shown in Figure 1A and Figure S2.

Bottom Line: We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100).Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease.Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging "cancer methylome". In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-beta (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

Show MeSH
Related in: MedlinePlus