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RUNX3 has an oncogenic role in head and neck cancer.

Tsunematsu T, Kudo Y, Iizuka S, Ogawa I, Fujita T, Kurihara H, Abiko Y, Takata T - PLoS ONE (2009)

Bottom Line: These findings were confirmed by RUNX3 knockdown.Moreover, RUNX3 expression was low due to the methylation of its promoter in normal oral epithelial cells.Our findings suggest that i) RUNX3 has an oncogenic role in HNSCC, ii) RUNX3 expression observed in HNSCC may be caused in part by demethylation during cancer development, and iii) RUNX3 expression can be a useful marker for predicting malignant behavior and the effect of chemotherapeutic drugs in HNSCC.

View Article: PubMed Central - PubMed

Affiliation: Division of Frontier Medical Science, Department of Oral and Maxillofacial Pathobiology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT

Background: Runt-related transcription factor 3 (RUNX3) is a tumor suppressor of cancer and appears to be an important component of the transforming growth factor-beta (TGF-ss)-induced tumor suppression pathway. Surprisingly, we found that RUNX3 expression level in head and neck squamous cell carcinoma (HNSCC) tissues, which is one of the most common types of human cancer, was higher than that in normal tissues by a previously published microarray dataset in our preliminary study. Therefore, here we examined the oncogenic role of RUNX3 in HNSCC.

Principal findings: Frequent RUNX3 expression and its correlation with malignant behavior were observed in HNSCC. Ectopic RUNX3 overexpression promoted cell growth and inhibited serum starvation-induced apoptosis and chemotherapeutic drug induced apoptosis in HNSCC cells. These findings were confirmed by RUNX3 knockdown. Moreover, RUNX3 overexpression enhanced tumorsphere formation. RUNX3 expression level was well correlated with the methylation status in HNSCC cells. Moreover, RUNX3 expression was low due to the methylation of its promoter in normal oral epithelial cells.

Conclusions/significance: Our findings suggest that i) RUNX3 has an oncogenic role in HNSCC, ii) RUNX3 expression observed in HNSCC may be caused in part by demethylation during cancer development, and iii) RUNX3 expression can be a useful marker for predicting malignant behavior and the effect of chemotherapeutic drugs in HNSCC.

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Related in: MedlinePlus

Methylation status in normal oral epithelial cells and HNSCC.A: RUNX3 expression was examined after 5-aza-2′-deoxycytidine (5-Aza) treatment. HSC4 cells were treated with medium containing 300 nM 5-aza-2′-deoxycytidine for 72 h. After treatment, cells were collected and examined the expression of RUNX3 mRNA by RT-PCR. B: RUNX3 CpG island and analyzed regions (No. 1–10) are shown as vertical bars. Transcriptional start site (TSS) is located within region No. 7. C: Methylation status of RUNX3 in HNSCC cell lines. Genomic DNA was extracted from HNSCC cell lines and was treated with bisulfite. Methylation status of promoter region (No. 1–10) was examined by methylation specific PCR method. D: The summary of methylation and expression status of RUNX3 in HNSCC cell lines. E: Expression of Runx3 in mouse tongue epithelium of embryo (upper panel) and adult (lower panel) mouse by immunohistochemistry. Tongue tissues of mouse embryos at embryonic day 15.5 and 10 weeks old BALB/c mice were used. F: RUNX3 mRNA expression was examined by RT-PCR in 4 primary cultured normal oral epithelial cells (#1–#4). HSC4 cell was used as a negative control and Ca9-22 cell was used as a positive control for RUNX3 expression. GAPDH was used as a control. G: RUNX3 expression was examined after 5-aza-2′-deoxycytidine (5-Aza) treatment. Primary cultured normal oral epithelial cells (#1 and #2) were treated with medium containing 300 nM 5-aza-2′-deoxycytidine for 72 h. After treatment, cells were collected and examined the expression of RUNX3 mRNA by RT-PCR. H: Genomic DNA was extracted from 4 primary cultured normal oral epithelial cells (#1–#4). Methylation status at region No. 5–8 was examined by methylation specific PCR method. I: The summary of methylation and expression status of RUNX3 in normal epithelial cells.
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pone-0005892-g003: Methylation status in normal oral epithelial cells and HNSCC.A: RUNX3 expression was examined after 5-aza-2′-deoxycytidine (5-Aza) treatment. HSC4 cells were treated with medium containing 300 nM 5-aza-2′-deoxycytidine for 72 h. After treatment, cells were collected and examined the expression of RUNX3 mRNA by RT-PCR. B: RUNX3 CpG island and analyzed regions (No. 1–10) are shown as vertical bars. Transcriptional start site (TSS) is located within region No. 7. C: Methylation status of RUNX3 in HNSCC cell lines. Genomic DNA was extracted from HNSCC cell lines and was treated with bisulfite. Methylation status of promoter region (No. 1–10) was examined by methylation specific PCR method. D: The summary of methylation and expression status of RUNX3 in HNSCC cell lines. E: Expression of Runx3 in mouse tongue epithelium of embryo (upper panel) and adult (lower panel) mouse by immunohistochemistry. Tongue tissues of mouse embryos at embryonic day 15.5 and 10 weeks old BALB/c mice were used. F: RUNX3 mRNA expression was examined by RT-PCR in 4 primary cultured normal oral epithelial cells (#1–#4). HSC4 cell was used as a negative control and Ca9-22 cell was used as a positive control for RUNX3 expression. GAPDH was used as a control. G: RUNX3 expression was examined after 5-aza-2′-deoxycytidine (5-Aza) treatment. Primary cultured normal oral epithelial cells (#1 and #2) were treated with medium containing 300 nM 5-aza-2′-deoxycytidine for 72 h. After treatment, cells were collected and examined the expression of RUNX3 mRNA by RT-PCR. H: Genomic DNA was extracted from 4 primary cultured normal oral epithelial cells (#1–#4). Methylation status at region No. 5–8 was examined by methylation specific PCR method. I: The summary of methylation and expression status of RUNX3 in normal epithelial cells.

Mentions: We asked how RUNX3 expression was caused by in HNSCC. Reduced expression of RUNX3 was frequently caused by CpG island hypermethylation in various types of cancer [9]. In fact, RUNX3 expression was observed in HSC4 cells without RUNX3 expression after 5-aza-2′-deoxycytidine treatment (Figure 3A). Homma et al. examined the methylation status of multiple regions of RUNX3 promoter CpG island (3478 bp) within the proximal promoter (No. 1–10) in gastric cancers and found that methylation at the region spanning the transcription start site (No. 5–8) is critical for loss of the RUNX3 expression (Figure 3B) [15]. To examine the RUNX3 methylation status in HNSCC, we analyzed the methylation of RUNX3 at all promoter regions (No. 1–10) by methylation-specific PCR on the panel of HNSCC cell lines (Figure 3C and 3D). HNSCC cells with RUNX3 expression showed unmethylation or partially methylation at No. 5–8 region (Figure 3D). HNSCC cells without RUNX3 expression showed fully methylation at No. 5 and No. 6 region. Thus, methylation status of the RUNX3 promoter region was well correlated with RUNX3 mRNA expression in HNSCC cell lines.


RUNX3 has an oncogenic role in head and neck cancer.

Tsunematsu T, Kudo Y, Iizuka S, Ogawa I, Fujita T, Kurihara H, Abiko Y, Takata T - PLoS ONE (2009)

Methylation status in normal oral epithelial cells and HNSCC.A: RUNX3 expression was examined after 5-aza-2′-deoxycytidine (5-Aza) treatment. HSC4 cells were treated with medium containing 300 nM 5-aza-2′-deoxycytidine for 72 h. After treatment, cells were collected and examined the expression of RUNX3 mRNA by RT-PCR. B: RUNX3 CpG island and analyzed regions (No. 1–10) are shown as vertical bars. Transcriptional start site (TSS) is located within region No. 7. C: Methylation status of RUNX3 in HNSCC cell lines. Genomic DNA was extracted from HNSCC cell lines and was treated with bisulfite. Methylation status of promoter region (No. 1–10) was examined by methylation specific PCR method. D: The summary of methylation and expression status of RUNX3 in HNSCC cell lines. E: Expression of Runx3 in mouse tongue epithelium of embryo (upper panel) and adult (lower panel) mouse by immunohistochemistry. Tongue tissues of mouse embryos at embryonic day 15.5 and 10 weeks old BALB/c mice were used. F: RUNX3 mRNA expression was examined by RT-PCR in 4 primary cultured normal oral epithelial cells (#1–#4). HSC4 cell was used as a negative control and Ca9-22 cell was used as a positive control for RUNX3 expression. GAPDH was used as a control. G: RUNX3 expression was examined after 5-aza-2′-deoxycytidine (5-Aza) treatment. Primary cultured normal oral epithelial cells (#1 and #2) were treated with medium containing 300 nM 5-aza-2′-deoxycytidine for 72 h. After treatment, cells were collected and examined the expression of RUNX3 mRNA by RT-PCR. H: Genomic DNA was extracted from 4 primary cultured normal oral epithelial cells (#1–#4). Methylation status at region No. 5–8 was examined by methylation specific PCR method. I: The summary of methylation and expression status of RUNX3 in normal epithelial cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2690822&req=5

pone-0005892-g003: Methylation status in normal oral epithelial cells and HNSCC.A: RUNX3 expression was examined after 5-aza-2′-deoxycytidine (5-Aza) treatment. HSC4 cells were treated with medium containing 300 nM 5-aza-2′-deoxycytidine for 72 h. After treatment, cells were collected and examined the expression of RUNX3 mRNA by RT-PCR. B: RUNX3 CpG island and analyzed regions (No. 1–10) are shown as vertical bars. Transcriptional start site (TSS) is located within region No. 7. C: Methylation status of RUNX3 in HNSCC cell lines. Genomic DNA was extracted from HNSCC cell lines and was treated with bisulfite. Methylation status of promoter region (No. 1–10) was examined by methylation specific PCR method. D: The summary of methylation and expression status of RUNX3 in HNSCC cell lines. E: Expression of Runx3 in mouse tongue epithelium of embryo (upper panel) and adult (lower panel) mouse by immunohistochemistry. Tongue tissues of mouse embryos at embryonic day 15.5 and 10 weeks old BALB/c mice were used. F: RUNX3 mRNA expression was examined by RT-PCR in 4 primary cultured normal oral epithelial cells (#1–#4). HSC4 cell was used as a negative control and Ca9-22 cell was used as a positive control for RUNX3 expression. GAPDH was used as a control. G: RUNX3 expression was examined after 5-aza-2′-deoxycytidine (5-Aza) treatment. Primary cultured normal oral epithelial cells (#1 and #2) were treated with medium containing 300 nM 5-aza-2′-deoxycytidine for 72 h. After treatment, cells were collected and examined the expression of RUNX3 mRNA by RT-PCR. H: Genomic DNA was extracted from 4 primary cultured normal oral epithelial cells (#1–#4). Methylation status at region No. 5–8 was examined by methylation specific PCR method. I: The summary of methylation and expression status of RUNX3 in normal epithelial cells.
Mentions: We asked how RUNX3 expression was caused by in HNSCC. Reduced expression of RUNX3 was frequently caused by CpG island hypermethylation in various types of cancer [9]. In fact, RUNX3 expression was observed in HSC4 cells without RUNX3 expression after 5-aza-2′-deoxycytidine treatment (Figure 3A). Homma et al. examined the methylation status of multiple regions of RUNX3 promoter CpG island (3478 bp) within the proximal promoter (No. 1–10) in gastric cancers and found that methylation at the region spanning the transcription start site (No. 5–8) is critical for loss of the RUNX3 expression (Figure 3B) [15]. To examine the RUNX3 methylation status in HNSCC, we analyzed the methylation of RUNX3 at all promoter regions (No. 1–10) by methylation-specific PCR on the panel of HNSCC cell lines (Figure 3C and 3D). HNSCC cells with RUNX3 expression showed unmethylation or partially methylation at No. 5–8 region (Figure 3D). HNSCC cells without RUNX3 expression showed fully methylation at No. 5 and No. 6 region. Thus, methylation status of the RUNX3 promoter region was well correlated with RUNX3 mRNA expression in HNSCC cell lines.

Bottom Line: These findings were confirmed by RUNX3 knockdown.Moreover, RUNX3 expression was low due to the methylation of its promoter in normal oral epithelial cells.Our findings suggest that i) RUNX3 has an oncogenic role in HNSCC, ii) RUNX3 expression observed in HNSCC may be caused in part by demethylation during cancer development, and iii) RUNX3 expression can be a useful marker for predicting malignant behavior and the effect of chemotherapeutic drugs in HNSCC.

View Article: PubMed Central - PubMed

Affiliation: Division of Frontier Medical Science, Department of Oral and Maxillofacial Pathobiology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT

Background: Runt-related transcription factor 3 (RUNX3) is a tumor suppressor of cancer and appears to be an important component of the transforming growth factor-beta (TGF-ss)-induced tumor suppression pathway. Surprisingly, we found that RUNX3 expression level in head and neck squamous cell carcinoma (HNSCC) tissues, which is one of the most common types of human cancer, was higher than that in normal tissues by a previously published microarray dataset in our preliminary study. Therefore, here we examined the oncogenic role of RUNX3 in HNSCC.

Principal findings: Frequent RUNX3 expression and its correlation with malignant behavior were observed in HNSCC. Ectopic RUNX3 overexpression promoted cell growth and inhibited serum starvation-induced apoptosis and chemotherapeutic drug induced apoptosis in HNSCC cells. These findings were confirmed by RUNX3 knockdown. Moreover, RUNX3 overexpression enhanced tumorsphere formation. RUNX3 expression level was well correlated with the methylation status in HNSCC cells. Moreover, RUNX3 expression was low due to the methylation of its promoter in normal oral epithelial cells.

Conclusions/significance: Our findings suggest that i) RUNX3 has an oncogenic role in HNSCC, ii) RUNX3 expression observed in HNSCC may be caused in part by demethylation during cancer development, and iii) RUNX3 expression can be a useful marker for predicting malignant behavior and the effect of chemotherapeutic drugs in HNSCC.

Show MeSH
Related in: MedlinePlus