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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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Immuno-expression of RUNX3 in HNSCC.A: Expression of RUNX3 was examined in skin epithelium (×100 and ×250, upper panel), normal oral mucosae (×100 and ×250, middle panel) and normal oral mucosae with infiltration of chronic inflammatory cells (×100 and ×250, lower panel). In skin epithelium, some of epidermal cells showed RUNX3 expression in its nuclei. In normal oral mucosal tissues, only a few epithelial cells in basal layer expressed RUNX3 in their nuclei, and most epithelial cells did not express RUNX3. In normal oral mucosae with infiltration of chronic inflammatory cells, lymphocytes infiltrating into oral mucosa showed RUNX3 expression. B: Expression of RUNX3 was examined in HNSCC cases (×100). Most cancer cells expressed RUNX3 in their nuclei.
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pone-0005892-g002: Immuno-expression of RUNX3 in HNSCC.A: Expression of RUNX3 was examined in skin epithelium (×100 and ×250, upper panel), normal oral mucosae (×100 and ×250, middle panel) and normal oral mucosae with infiltration of chronic inflammatory cells (×100 and ×250, lower panel). In skin epithelium, some of epidermal cells showed RUNX3 expression in its nuclei. In normal oral mucosal tissues, only a few epithelial cells in basal layer expressed RUNX3 in their nuclei, and most epithelial cells did not express RUNX3. In normal oral mucosae with infiltration of chronic inflammatory cells, lymphocytes infiltrating into oral mucosa showed RUNX3 expression. B: Expression of RUNX3 was examined in HNSCC cases (×100). Most cancer cells expressed RUNX3 in their nuclei.

Mentions: Genomic DNA from cells or tissues was extracted using the DNeasy Kit (Qiagen, Hilden, Germany). Fifty micro liters of the supernatant were used directly as a source of DNA for sodium bisulfite treatment. DNA samples were treated with bisulfite to convert all unmethylated cytosines to uracils whilst leaving methylated cytosines unaffected. DNA was denatured by NaOH (final concentration, 0.2 M) for 10 min at 37°C. Thirty µl of 10 mM hydroquinone (Sigma) and 520 µl of 3 M sodium bisulfite (Sigma) at pH 5.0 both freshly prepared, were added and mixed, and samples were incubated at 50°C for 16 h. Modified DNA was purified using Wizard DNA purification resin (Promega) and eluted into 50 µl of water. Modification was completed by NaOH (final concentration, 0.3 M) treatment for 5 min at room temperature, followed by ethanol precipitation. Modified DNAs were amplified in a 20 µL reaction volume containing 2 µL 10×PCR buffer with 15 mM MgCl2, 4 µL 5xQ-Solution, 10 pM of each primer, 0.2 mM dNTPs and 0.75 U Taq polymerase (HotStar Taq DNA polymerase; Qiagen, Hilden, Germany). After the mixture was heated at 95°C for 15 min, PCR was performed in a thermal cycler (GeneAmp 2400; PE Applied Biosystems, Foster City, CA, USA) for 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 60 sec, and extension at 72°C for 60 sec, followed by a final 10 min extension at 72°C. The PCR products were separated on a 8% non-denaturing polyacrylamide gel. RUNX3 CpG island and analyzed regions (No. 1–10) are shown in Figure 2B. The primer sets used in this study were listed in Table S1 (GenBank accession number AL023096) [15].


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)

Immuno-expression of RUNX3 in HNSCC.A: Expression of RUNX3 was examined in skin epithelium (×100 and ×250, upper panel), normal oral mucosae (×100 and ×250, middle panel) and normal oral mucosae with infiltration of chronic inflammatory cells (×100 and ×250, lower panel). In skin epithelium, some of epidermal cells showed RUNX3 expression in its nuclei. In normal oral mucosal tissues, only a few epithelial cells in basal layer expressed RUNX3 in their nuclei, and most epithelial cells did not express RUNX3. In normal oral mucosae with infiltration of chronic inflammatory cells, lymphocytes infiltrating into oral mucosa showed RUNX3 expression. B: Expression of RUNX3 was examined in HNSCC cases (×100). Most cancer cells expressed RUNX3 in their nuclei.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005892-g002: Immuno-expression of RUNX3 in HNSCC.A: Expression of RUNX3 was examined in skin epithelium (×100 and ×250, upper panel), normal oral mucosae (×100 and ×250, middle panel) and normal oral mucosae with infiltration of chronic inflammatory cells (×100 and ×250, lower panel). In skin epithelium, some of epidermal cells showed RUNX3 expression in its nuclei. In normal oral mucosal tissues, only a few epithelial cells in basal layer expressed RUNX3 in their nuclei, and most epithelial cells did not express RUNX3. In normal oral mucosae with infiltration of chronic inflammatory cells, lymphocytes infiltrating into oral mucosa showed RUNX3 expression. B: Expression of RUNX3 was examined in HNSCC cases (×100). Most cancer cells expressed RUNX3 in their nuclei.
Mentions: Genomic DNA from cells or tissues was extracted using the DNeasy Kit (Qiagen, Hilden, Germany). Fifty micro liters of the supernatant were used directly as a source of DNA for sodium bisulfite treatment. DNA samples were treated with bisulfite to convert all unmethylated cytosines to uracils whilst leaving methylated cytosines unaffected. DNA was denatured by NaOH (final concentration, 0.2 M) for 10 min at 37°C. Thirty µl of 10 mM hydroquinone (Sigma) and 520 µl of 3 M sodium bisulfite (Sigma) at pH 5.0 both freshly prepared, were added and mixed, and samples were incubated at 50°C for 16 h. Modified DNA was purified using Wizard DNA purification resin (Promega) and eluted into 50 µl of water. Modification was completed by NaOH (final concentration, 0.3 M) treatment for 5 min at room temperature, followed by ethanol precipitation. Modified DNAs were amplified in a 20 µL reaction volume containing 2 µL 10×PCR buffer with 15 mM MgCl2, 4 µL 5xQ-Solution, 10 pM of each primer, 0.2 mM dNTPs and 0.75 U Taq polymerase (HotStar Taq DNA polymerase; Qiagen, Hilden, Germany). After the mixture was heated at 95°C for 15 min, PCR was performed in a thermal cycler (GeneAmp 2400; PE Applied Biosystems, Foster City, CA, USA) for 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 60 sec, and extension at 72°C for 60 sec, followed by a final 10 min extension at 72°C. The PCR products were separated on a 8% non-denaturing polyacrylamide gel. RUNX3 CpG island and analyzed regions (No. 1–10) are shown in Figure 2B. The primer sets used in this study were listed in Table S1 (GenBank accession number AL023096) [15].

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