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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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Cellular function of OSMR.A, Analysis of OSMR promoter activity by luciferase reporter assay in OSMR-positive (HCT116 and HEK293) and - negative (SW480) cells. The promoter construct was pre-treated with or without SssI methylase for 8 hrs before transfection. The highest activity was detected in HEK293 cells. Data are expressed as fold increase over pGL3-basic activity. Experiments were done in triplicate, and values indicate means±SD. Mean values are presented. B, An siRNA pool targeting OSMR and non-targeting control were transiently transfected into HCT116, and cells were treated with rhOSM (50 ng/ml) (left). HCT116 cells were treated with or without Stat3 inhibitor peptide (Stat3 Inh, 100 μM) acting as a highly selective, potent blocker of Stat3 activation (right). Cell growth was determined by MTT assay. Data are expressed as absorbance at 570 nm. Experiments were done in triplicate, and values indicate means±SD. *, P<0.05. C, 5-Aza-dC (5 μM) was pre-treated for 48 hrs in SW480 and DLD-1 cells, and then co-treated with rhOSM for 48 hrs. Cell growth was determined by MTT assay. *, 5-Aza-dC-treated cells compared to untreated control (P<0.05); #, 5-Aza-dC/rhOSM- treated cells compared to 5-Aza-dC treatment alone (P<0.05). D, The phosphorylation of Stat3 and Erk in response to rhOSM (50 ng/ml) was analyzed by Western blotting in HCT116 and SW480 cell lines. rhOSM increased phosphorylated Stat3 and Erk in HCT116 cells but not in SW480 cells (which lack LIFR-see E below). Equal protein loading was monitored by total Stat3, Erk and β-actin evaluation in the samples. Re-activation of OSMR by 5-Aza-dC treatment was determined on SW480 and DLD-1 cells by flow cytometry (Figure S6). E. Expression of LIFR and gp130 was examined in CRC cell lines by RT-PCR. LIFR was expressed in HCT116 cells but silenced in most other CRC cell lines tested. gp130, the OSMR heterodimer, was ubiquitously expressed in all cell lines tested. Promoter methylation status of these two latter genes was not examined.
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pone-0006555-g005: Cellular function of OSMR.A, Analysis of OSMR promoter activity by luciferase reporter assay in OSMR-positive (HCT116 and HEK293) and - negative (SW480) cells. The promoter construct was pre-treated with or without SssI methylase for 8 hrs before transfection. The highest activity was detected in HEK293 cells. Data are expressed as fold increase over pGL3-basic activity. Experiments were done in triplicate, and values indicate means±SD. Mean values are presented. B, An siRNA pool targeting OSMR and non-targeting control were transiently transfected into HCT116, and cells were treated with rhOSM (50 ng/ml) (left). HCT116 cells were treated with or without Stat3 inhibitor peptide (Stat3 Inh, 100 μM) acting as a highly selective, potent blocker of Stat3 activation (right). Cell growth was determined by MTT assay. Data are expressed as absorbance at 570 nm. Experiments were done in triplicate, and values indicate means±SD. *, P<0.05. C, 5-Aza-dC (5 μM) was pre-treated for 48 hrs in SW480 and DLD-1 cells, and then co-treated with rhOSM for 48 hrs. Cell growth was determined by MTT assay. *, 5-Aza-dC-treated cells compared to untreated control (P<0.05); #, 5-Aza-dC/rhOSM- treated cells compared to 5-Aza-dC treatment alone (P<0.05). D, The phosphorylation of Stat3 and Erk in response to rhOSM (50 ng/ml) was analyzed by Western blotting in HCT116 and SW480 cell lines. rhOSM increased phosphorylated Stat3 and Erk in HCT116 cells but not in SW480 cells (which lack LIFR-see E below). Equal protein loading was monitored by total Stat3, Erk and β-actin evaluation in the samples. Re-activation of OSMR by 5-Aza-dC treatment was determined on SW480 and DLD-1 cells by flow cytometry (Figure S6). E. Expression of LIFR and gp130 was examined in CRC cell lines by RT-PCR. LIFR was expressed in HCT116 cells but silenced in most other CRC cell lines tested. gp130, the OSMR heterodimer, was ubiquitously expressed in all cell lines tested. Promoter methylation status of these two latter genes was not examined.

Mentions: To investigate the role of DNA methylation in regulation of OSMR expression, we transfected a pGL3-OSMR-Pro2-Luciferase construct into three cell lines; a OSMR-negative cell line, SW480, and two OSMR-positive cell lines, HCT116 and HEK293. The construct was treated with or without SssI methylase before transfection. Activity of the OSMR promoter was not detected in SW480, but a high level of promoter activity was detected in HCT116 and HEK293 cells (Fig. 5A). Induction of CpG methylation with SssI methylase decreased the activity to minimal levels.


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)

Cellular function of OSMR.A, Analysis of OSMR promoter activity by luciferase reporter assay in OSMR-positive (HCT116 and HEK293) and - negative (SW480) cells. The promoter construct was pre-treated with or without SssI methylase for 8 hrs before transfection. The highest activity was detected in HEK293 cells. Data are expressed as fold increase over pGL3-basic activity. Experiments were done in triplicate, and values indicate means±SD. Mean values are presented. B, An siRNA pool targeting OSMR and non-targeting control were transiently transfected into HCT116, and cells were treated with rhOSM (50 ng/ml) (left). HCT116 cells were treated with or without Stat3 inhibitor peptide (Stat3 Inh, 100 μM) acting as a highly selective, potent blocker of Stat3 activation (right). Cell growth was determined by MTT assay. Data are expressed as absorbance at 570 nm. Experiments were done in triplicate, and values indicate means±SD. *, P<0.05. C, 5-Aza-dC (5 μM) was pre-treated for 48 hrs in SW480 and DLD-1 cells, and then co-treated with rhOSM for 48 hrs. Cell growth was determined by MTT assay. *, 5-Aza-dC-treated cells compared to untreated control (P<0.05); #, 5-Aza-dC/rhOSM- treated cells compared to 5-Aza-dC treatment alone (P<0.05). D, The phosphorylation of Stat3 and Erk in response to rhOSM (50 ng/ml) was analyzed by Western blotting in HCT116 and SW480 cell lines. rhOSM increased phosphorylated Stat3 and Erk in HCT116 cells but not in SW480 cells (which lack LIFR-see E below). Equal protein loading was monitored by total Stat3, Erk and β-actin evaluation in the samples. Re-activation of OSMR by 5-Aza-dC treatment was determined on SW480 and DLD-1 cells by flow cytometry (Figure S6). E. Expression of LIFR and gp130 was examined in CRC cell lines by RT-PCR. LIFR was expressed in HCT116 cells but silenced in most other CRC cell lines tested. gp130, the OSMR heterodimer, was ubiquitously expressed in all cell lines tested. Promoter methylation status of these two latter genes was not examined.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2717211&req=5

pone-0006555-g005: Cellular function of OSMR.A, Analysis of OSMR promoter activity by luciferase reporter assay in OSMR-positive (HCT116 and HEK293) and - negative (SW480) cells. The promoter construct was pre-treated with or without SssI methylase for 8 hrs before transfection. The highest activity was detected in HEK293 cells. Data are expressed as fold increase over pGL3-basic activity. Experiments were done in triplicate, and values indicate means±SD. Mean values are presented. B, An siRNA pool targeting OSMR and non-targeting control were transiently transfected into HCT116, and cells were treated with rhOSM (50 ng/ml) (left). HCT116 cells were treated with or without Stat3 inhibitor peptide (Stat3 Inh, 100 μM) acting as a highly selective, potent blocker of Stat3 activation (right). Cell growth was determined by MTT assay. Data are expressed as absorbance at 570 nm. Experiments were done in triplicate, and values indicate means±SD. *, P<0.05. C, 5-Aza-dC (5 μM) was pre-treated for 48 hrs in SW480 and DLD-1 cells, and then co-treated with rhOSM for 48 hrs. Cell growth was determined by MTT assay. *, 5-Aza-dC-treated cells compared to untreated control (P<0.05); #, 5-Aza-dC/rhOSM- treated cells compared to 5-Aza-dC treatment alone (P<0.05). D, The phosphorylation of Stat3 and Erk in response to rhOSM (50 ng/ml) was analyzed by Western blotting in HCT116 and SW480 cell lines. rhOSM increased phosphorylated Stat3 and Erk in HCT116 cells but not in SW480 cells (which lack LIFR-see E below). Equal protein loading was monitored by total Stat3, Erk and β-actin evaluation in the samples. Re-activation of OSMR by 5-Aza-dC treatment was determined on SW480 and DLD-1 cells by flow cytometry (Figure S6). E. Expression of LIFR and gp130 was examined in CRC cell lines by RT-PCR. LIFR was expressed in HCT116 cells but silenced in most other CRC cell lines tested. gp130, the OSMR heterodimer, was ubiquitously expressed in all cell lines tested. Promoter methylation status of these two latter genes was not examined.
Mentions: To investigate the role of DNA methylation in regulation of OSMR expression, we transfected a pGL3-OSMR-Pro2-Luciferase construct into three cell lines; a OSMR-negative cell line, SW480, and two OSMR-positive cell lines, HCT116 and HEK293. The construct was treated with or without SssI methylase before transfection. Activity of the OSMR promoter was not detected in SW480, but a high level of promoter activity was detected in HCT116 and HEK293 cells (Fig. 5A). Induction of CpG methylation with SssI methylase decreased the activity to minimal levels.

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