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Context-dependent activation of Wnt signaling by tumor suppressor RUNX3 in gastric cancer cells.

Ju X, Ishikawa TO, Naka K, Ito K, Ito Y, Oshima M - Cancer Sci. (2014)

Bottom Line: Here, we confirmed that RUNX3 suppressed Wnt signaling activity in several gastric cancer cell lines; however, we found that RUNX3 increased the Wnt signaling activity in KatoIII and SNU668 gastric cancer cells.As found previously, RUNX3 also binds to TCF4 and β-catenin in KatoIII cells, suggesting that these molecules form a ternary complex.These results indicate that RUNX3 can either suppress or activate the Wnt signaling pathway through its binding to the TCF4/β-catenin complex by cell context-dependent mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan.

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RUNX3-induced Wnt signaling activation. (a) Western blotting of the RUNX3 expression in gastric cancer cells. (b) Relative luciferase activity of TOPflash/FOPflash in the respective gastric cancer cells transfected with RUNX3 or R122C mutant RUNX3 vector to the control vector-transfected cells. (c) Luciferase activity of the TOPflash and FOPflash in KatoIII-R3 cells and siRNA-transfected KatoIII-R3 cells relative to the level in the parental KatoIII cells. (d) Relative luciferase activity of TOPflash/FOPflash in KatoIII cells transfected with RUNX3 expression vector. (e) Relative expression levels of the Wnt target genes, SOX4 and Axin2, detected by RT-PCR in the RUNX3 vector-transfected cells (red) or RUNX3 siRNA-transfected cells (blue) to the control level. (f) Relative luciferase activity of TOPflash/FOPflash in AZ521 and MKN45 cells transfected with RUNX3 siRNA to the control level. (b–f) Bar graphs are shown as mean ± SD. *P < 0.05 versus control levels otherwise indicated.
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fig01: RUNX3-induced Wnt signaling activation. (a) Western blotting of the RUNX3 expression in gastric cancer cells. (b) Relative luciferase activity of TOPflash/FOPflash in the respective gastric cancer cells transfected with RUNX3 or R122C mutant RUNX3 vector to the control vector-transfected cells. (c) Luciferase activity of the TOPflash and FOPflash in KatoIII-R3 cells and siRNA-transfected KatoIII-R3 cells relative to the level in the parental KatoIII cells. (d) Relative luciferase activity of TOPflash/FOPflash in KatoIII cells transfected with RUNX3 expression vector. (e) Relative expression levels of the Wnt target genes, SOX4 and Axin2, detected by RT-PCR in the RUNX3 vector-transfected cells (red) or RUNX3 siRNA-transfected cells (blue) to the control level. (f) Relative luciferase activity of TOPflash/FOPflash in AZ521 and MKN45 cells transfected with RUNX3 siRNA to the control level. (b–f) Bar graphs are shown as mean ± SD. *P < 0.05 versus control levels otherwise indicated.

Mentions: RUNX3 expression was detected by western blotting in AZ521 and MKN45 cells, while it was not detected in other gastric cancer cell lines (Fig. 1a). Consistently, high levels of RUNX3 mRNA were detected in AZ521 and MKN45 cells by RT-PCR (Fig. S1). We transiently transfected the RUNX3 expression vector to all gastric cell lines, and examined Wnt signaling activity by luciferase reporter analysis. The Wnt signaling activity was significantly decreased in SNU216, SNU601, SNU638 and SNU719 cells, which was consistent with the previous results.(12) However, RUNX3 expression increased Wnt signaling activity in KatoIII and SNU668 cells (Fig. 1b). Importantly, the R122C mutant form of RUNX3 that is defective in the RUNX3 function did not change Wnt signaling activity in these cells.(1,6) The KatoIII-R3, stable RUNX3-expressing cells (Fig. S2) also exhibited an increased luciferase activity, which was suppressed by RUNX3 siRNA transfection (Fig. 1c). Moreover, Wnt activation levels increased gradually in accordance with the amount of the RUNX3 expression vector (Fig. 1d). Consistently, the expression levels of Wnt target genes, Sox4 and Axin2, increased significantly in KatoIII and SNU668 cells by RUNX3 expression (Fig. 1e).


Context-dependent activation of Wnt signaling by tumor suppressor RUNX3 in gastric cancer cells.

Ju X, Ishikawa TO, Naka K, Ito K, Ito Y, Oshima M - Cancer Sci. (2014)

RUNX3-induced Wnt signaling activation. (a) Western blotting of the RUNX3 expression in gastric cancer cells. (b) Relative luciferase activity of TOPflash/FOPflash in the respective gastric cancer cells transfected with RUNX3 or R122C mutant RUNX3 vector to the control vector-transfected cells. (c) Luciferase activity of the TOPflash and FOPflash in KatoIII-R3 cells and siRNA-transfected KatoIII-R3 cells relative to the level in the parental KatoIII cells. (d) Relative luciferase activity of TOPflash/FOPflash in KatoIII cells transfected with RUNX3 expression vector. (e) Relative expression levels of the Wnt target genes, SOX4 and Axin2, detected by RT-PCR in the RUNX3 vector-transfected cells (red) or RUNX3 siRNA-transfected cells (blue) to the control level. (f) Relative luciferase activity of TOPflash/FOPflash in AZ521 and MKN45 cells transfected with RUNX3 siRNA to the control level. (b–f) Bar graphs are shown as mean ± SD. *P < 0.05 versus control levels otherwise indicated.
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fig01: RUNX3-induced Wnt signaling activation. (a) Western blotting of the RUNX3 expression in gastric cancer cells. (b) Relative luciferase activity of TOPflash/FOPflash in the respective gastric cancer cells transfected with RUNX3 or R122C mutant RUNX3 vector to the control vector-transfected cells. (c) Luciferase activity of the TOPflash and FOPflash in KatoIII-R3 cells and siRNA-transfected KatoIII-R3 cells relative to the level in the parental KatoIII cells. (d) Relative luciferase activity of TOPflash/FOPflash in KatoIII cells transfected with RUNX3 expression vector. (e) Relative expression levels of the Wnt target genes, SOX4 and Axin2, detected by RT-PCR in the RUNX3 vector-transfected cells (red) or RUNX3 siRNA-transfected cells (blue) to the control level. (f) Relative luciferase activity of TOPflash/FOPflash in AZ521 and MKN45 cells transfected with RUNX3 siRNA to the control level. (b–f) Bar graphs are shown as mean ± SD. *P < 0.05 versus control levels otherwise indicated.
Mentions: RUNX3 expression was detected by western blotting in AZ521 and MKN45 cells, while it was not detected in other gastric cancer cell lines (Fig. 1a). Consistently, high levels of RUNX3 mRNA were detected in AZ521 and MKN45 cells by RT-PCR (Fig. S1). We transiently transfected the RUNX3 expression vector to all gastric cell lines, and examined Wnt signaling activity by luciferase reporter analysis. The Wnt signaling activity was significantly decreased in SNU216, SNU601, SNU638 and SNU719 cells, which was consistent with the previous results.(12) However, RUNX3 expression increased Wnt signaling activity in KatoIII and SNU668 cells (Fig. 1b). Importantly, the R122C mutant form of RUNX3 that is defective in the RUNX3 function did not change Wnt signaling activity in these cells.(1,6) The KatoIII-R3, stable RUNX3-expressing cells (Fig. S2) also exhibited an increased luciferase activity, which was suppressed by RUNX3 siRNA transfection (Fig. 1c). Moreover, Wnt activation levels increased gradually in accordance with the amount of the RUNX3 expression vector (Fig. 1d). Consistently, the expression levels of Wnt target genes, Sox4 and Axin2, increased significantly in KatoIII and SNU668 cells by RUNX3 expression (Fig. 1e).

Bottom Line: Here, we confirmed that RUNX3 suppressed Wnt signaling activity in several gastric cancer cell lines; however, we found that RUNX3 increased the Wnt signaling activity in KatoIII and SNU668 gastric cancer cells.As found previously, RUNX3 also binds to TCF4 and β-catenin in KatoIII cells, suggesting that these molecules form a ternary complex.These results indicate that RUNX3 can either suppress or activate the Wnt signaling pathway through its binding to the TCF4/β-catenin complex by cell context-dependent mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan.

Show MeSH
Related in: MedlinePlus