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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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RUNX3 overexpression promoted cell growth and inhibited serum starvation induced apoptosis.A: Generation of RUNX3 overexpressing cells. The RUNX3/pcDNA3 plasmid or the vector alone was introduced into HSC3 cells, and the stable pool clones were obtained by G418 selection for 2 weeks. Exogenous expression of RUNX3 mRNA and protein was examined by RT-PCR and Western blot analysis (WB). Cul1 was used as a loading control. B: The graph shows cell growth of RUNX3 overexpressing and control HSC3 cells. Cells were plated on 24 well plates, and trypsinized cells were counted by Cell Counter (Coulter Z1) at 0, 2, 4 and 6 day. C: RUNX3 overexpression inhibited the serum starvation induced apoptosis. Cells were incubated for 0, 48 and 96 hours after serum starvation and fixed in 70% ethanol. Cell cycle distribution was determined by DNA content analysis after propidium iodide staining using a flow cytometer. For each sample, 20,000 events were stored. We performed two independent experiments. D: Flow cytometric analysis of Annexin V and propidium iodide staining in control and RUNX3 overexpressing cells after serum starvation for 96 h. We performed two independent experiments.
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pone-0005892-g004: RUNX3 overexpression promoted cell growth and inhibited serum starvation induced apoptosis.A: Generation of RUNX3 overexpressing cells. The RUNX3/pcDNA3 plasmid or the vector alone was introduced into HSC3 cells, and the stable pool clones were obtained by G418 selection for 2 weeks. Exogenous expression of RUNX3 mRNA and protein was examined by RT-PCR and Western blot analysis (WB). Cul1 was used as a loading control. B: The graph shows cell growth of RUNX3 overexpressing and control HSC3 cells. Cells were plated on 24 well plates, and trypsinized cells were counted by Cell Counter (Coulter Z1) at 0, 2, 4 and 6 day. C: RUNX3 overexpression inhibited the serum starvation induced apoptosis. Cells were incubated for 0, 48 and 96 hours after serum starvation and fixed in 70% ethanol. Cell cycle distribution was determined by DNA content analysis after propidium iodide staining using a flow cytometer. For each sample, 20,000 events were stored. We performed two independent experiments. D: Flow cytometric analysis of Annexin V and propidium iodide staining in control and RUNX3 overexpressing cells after serum starvation for 96 h. We performed two independent experiments.

Mentions: To know the role of RUNX3 in HNSCC, we stably transfected an expression vector of FLAG-RUNX3 into HSC3 cells with lower expression of RUNX3. We obtained stably RUNX3 overexpressing cells (Figure 4A). Interestingly, RUNX3 overexpression enhanced cell growth (Figure 4B). At 6day, number of RUNX3 overexpressing cells was remarkably higher than that of control cells. Although we examined migration and invasion, RUNX3 overexpression did not promote migration and invasion (Figure S2). Moreover, increased population corresponding to sub-G1 after serum starvation was observed only in control cells, but not in RUNX3 overexpressing cells (Figure 4C). To demonstrate whether this increment of sub-G1 detected in control cells after serum starvation is due to apoptosis, the level of apoptosis was assessed by using annexin V-FITC/propidium iodide assays (Figure 4D). Annexin V/propidium iodide double-positive cells were observed in 1.8% of RUNX3 overexpressing cells, while in 21.7% of control cells, indicating that RUNX3 overexpression inhibited serum starvation induced apoptosis. To confirm these phenotypes, we examined the knockdown of RUNX3 in Ca9-22 cells, which showed RUNX3 overexpression and were resistant to serum starvation-induced apoptosis. For knockdown of RUNX3, we used three different siRNAs (si-RUNX3-1, si-RUNX3-2 and si-RUNX3-3) and their cocktail. RUNX3 expression was remarkably silenced by si-RUNX3-1 (Figure S3A). Therefore, we used si-RUNX3-1 for the following studies. RUNX3 siRNA transfection reduced the expression of RUNX3 mRNA and protein in Ca9-22 cells (Figure 5A). We examined if siRNA induced a non-specific interferon stress response. RUNX3 siRNA treatment did not induce classic interferon-responsive genes, OAS1 and ISG54 mRNAs, indicating that RUNX3 siRNA treatment did not induce significant interferon response (Figure S3B). As we expected, RUNX3 siRNA inhibited the cell growth (Figure 5B). At 6day, number of RUNX3 siRNA treated cells was remarkably lower than that of control cells. Moreover, RUNX3 siRNA increased the population corresponding to sub-G1 after serum starvation (Figure 5C). Annexin V/propidium iodide double-positive cells were observed in 10.5% of control cells, while in 18.1% of RUNX3 knockdown cells (Figure 5D).


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)

RUNX3 overexpression promoted cell growth and inhibited serum starvation induced apoptosis.A: Generation of RUNX3 overexpressing cells. The RUNX3/pcDNA3 plasmid or the vector alone was introduced into HSC3 cells, and the stable pool clones were obtained by G418 selection for 2 weeks. Exogenous expression of RUNX3 mRNA and protein was examined by RT-PCR and Western blot analysis (WB). Cul1 was used as a loading control. B: The graph shows cell growth of RUNX3 overexpressing and control HSC3 cells. Cells were plated on 24 well plates, and trypsinized cells were counted by Cell Counter (Coulter Z1) at 0, 2, 4 and 6 day. C: RUNX3 overexpression inhibited the serum starvation induced apoptosis. Cells were incubated for 0, 48 and 96 hours after serum starvation and fixed in 70% ethanol. Cell cycle distribution was determined by DNA content analysis after propidium iodide staining using a flow cytometer. For each sample, 20,000 events were stored. We performed two independent experiments. D: Flow cytometric analysis of Annexin V and propidium iodide staining in control and RUNX3 overexpressing cells after serum starvation for 96 h. We performed two independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690822&req=5

pone-0005892-g004: RUNX3 overexpression promoted cell growth and inhibited serum starvation induced apoptosis.A: Generation of RUNX3 overexpressing cells. The RUNX3/pcDNA3 plasmid or the vector alone was introduced into HSC3 cells, and the stable pool clones were obtained by G418 selection for 2 weeks. Exogenous expression of RUNX3 mRNA and protein was examined by RT-PCR and Western blot analysis (WB). Cul1 was used as a loading control. B: The graph shows cell growth of RUNX3 overexpressing and control HSC3 cells. Cells were plated on 24 well plates, and trypsinized cells were counted by Cell Counter (Coulter Z1) at 0, 2, 4 and 6 day. C: RUNX3 overexpression inhibited the serum starvation induced apoptosis. Cells were incubated for 0, 48 and 96 hours after serum starvation and fixed in 70% ethanol. Cell cycle distribution was determined by DNA content analysis after propidium iodide staining using a flow cytometer. For each sample, 20,000 events were stored. We performed two independent experiments. D: Flow cytometric analysis of Annexin V and propidium iodide staining in control and RUNX3 overexpressing cells after serum starvation for 96 h. We performed two independent experiments.
Mentions: To know the role of RUNX3 in HNSCC, we stably transfected an expression vector of FLAG-RUNX3 into HSC3 cells with lower expression of RUNX3. We obtained stably RUNX3 overexpressing cells (Figure 4A). Interestingly, RUNX3 overexpression enhanced cell growth (Figure 4B). At 6day, number of RUNX3 overexpressing cells was remarkably higher than that of control cells. Although we examined migration and invasion, RUNX3 overexpression did not promote migration and invasion (Figure S2). Moreover, increased population corresponding to sub-G1 after serum starvation was observed only in control cells, but not in RUNX3 overexpressing cells (Figure 4C). To demonstrate whether this increment of sub-G1 detected in control cells after serum starvation is due to apoptosis, the level of apoptosis was assessed by using annexin V-FITC/propidium iodide assays (Figure 4D). Annexin V/propidium iodide double-positive cells were observed in 1.8% of RUNX3 overexpressing cells, while in 21.7% of control cells, indicating that RUNX3 overexpression inhibited serum starvation induced apoptosis. To confirm these phenotypes, we examined the knockdown of RUNX3 in Ca9-22 cells, which showed RUNX3 overexpression and were resistant to serum starvation-induced apoptosis. For knockdown of RUNX3, we used three different siRNAs (si-RUNX3-1, si-RUNX3-2 and si-RUNX3-3) and their cocktail. RUNX3 expression was remarkably silenced by si-RUNX3-1 (Figure S3A). Therefore, we used si-RUNX3-1 for the following studies. RUNX3 siRNA transfection reduced the expression of RUNX3 mRNA and protein in Ca9-22 cells (Figure 5A). We examined if siRNA induced a non-specific interferon stress response. RUNX3 siRNA treatment did not induce classic interferon-responsive genes, OAS1 and ISG54 mRNAs, indicating that RUNX3 siRNA treatment did not induce significant interferon response (Figure S3B). As we expected, RUNX3 siRNA inhibited the cell growth (Figure 5B). At 6day, number of RUNX3 siRNA treated cells was remarkably lower than that of control cells. Moreover, RUNX3 siRNA increased the population corresponding to sub-G1 after serum starvation (Figure 5C). Annexin V/propidium iodide double-positive cells were observed in 10.5% of control cells, while in 18.1% of RUNX3 knockdown cells (Figure 5D).

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