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NDRG2 expression decreases with tumor stages and regulates TCF/beta-catenin signaling in human colon carcinoma.

Kim YJ, Yoon SY, Kim JT, Song EY, Lee HG, Son HJ, Kim SY, Cho D, Choi I, Kim JH, Kim JW - Carcinogenesis (2009)

Bottom Line: SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid.TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis.Intracellular beta-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of beta-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea.

ABSTRACT
NDRG (N-Myc downstream-regulated gene)-2 is a member of the NDRG family. Although it has been suggested that NDRG2 is involved in cellular differentiation and tumor suppression, its intracellular signal and regulatory mechanism are not well known. Here, we show the differential expression of NDRG2 in human colon carcinoma cell lines and tissues by reverse transcription-polymerase chain reaction and immunohistochemical analyses with monoclonal antibody against NDRG2. NDRG2 was strongly expressed in normal colonic mucosa and colonic adenomatous tissues (25 of 25) but not in all invasive cancer tissues [44 of 99 (44%)]. Most distinctive results indicated that the high expression level of NDRG2 has a positive correlation with tumor differentiation and inverse correlation with tumor invasion depth and Dukes' stage of colon adenocarcinoma. To investigate the roles of NDRG2 in tumorigenesis, we used in vitro cell culture system. SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid. TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis. Intracellular beta-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of beta-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function. Our results suggest that NDRG2 might play a pivotal role as a potent tumor suppressor by the attenuation of TCF/beta-catenin signaling for the maintenance of healthy colon tissues.

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NDRG2 regulates the phosphorylation of GSK-3β. (A) NDRG2 introduction attenuated the increase of TCF/LEF activity following LiCl or β-catenin treatment. (B) Mutant NDRG2, NDRG2T334A and NDRG2Δ302 showed no effect on the transcription activity of TCF/LEF. (C) SW620/N was treated with NDRG2 siRNA. The reduction of NDRG2 mRNA and protein levels by siRNA was confirmed by RT–PCR and western blot analyses. The protein level of β-catenin was increased in the NDRG2 siRNA-introduced cells. (D) The suppression of LiCl-induced TCF activation by NDRG2 presented the possible function of NDRG2 in the regulation of GSK-3β activity, and the inhibitory phosphorylation of GSK-3β was reduced by NDRG2 overexpression. (E) Target genes of TCF/LEF transcription factor were downregulated following NDRG2 introduction.
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fig4: NDRG2 regulates the phosphorylation of GSK-3β. (A) NDRG2 introduction attenuated the increase of TCF/LEF activity following LiCl or β-catenin treatment. (B) Mutant NDRG2, NDRG2T334A and NDRG2Δ302 showed no effect on the transcription activity of TCF/LEF. (C) SW620/N was treated with NDRG2 siRNA. The reduction of NDRG2 mRNA and protein levels by siRNA was confirmed by RT–PCR and western blot analyses. The protein level of β-catenin was increased in the NDRG2 siRNA-introduced cells. (D) The suppression of LiCl-induced TCF activation by NDRG2 presented the possible function of NDRG2 in the regulation of GSK-3β activity, and the inhibitory phosphorylation of GSK-3β was reduced by NDRG2 overexpression. (E) Target genes of TCF/LEF transcription factor were downregulated following NDRG2 introduction.

Mentions: Protein stability of β-catenin is known to be regulated by the kinase activity of GSK-3β that seemed to be affected by NDRG2 function. TCF/LEF activity in SW620 cells was enhanced by treatment with LiCl, a well-known GSK-3β inhibitor, as well as by cotransfection of β-catenin. NDRG2-induced decrease of TCF/LEF activity was also detected under these enhanced conditions (Figure 4A). The data that NDRG2 attenuated the increase of TCF/LEF activity following LiCl treatment support the notion that NDRG2 may regulate the kinase activity of GSK-3β. Mutants NDRG2, NDRG2T334A, NDRG2T334D and NDRG2Δ302 showed no effect on the transcription activity of TCF/LEF (Figure 4B). Otherwise, as the expression of NDRG2 was downregulated by NDRG2 siRNA, the protein level of β-catenin was increased (Figure 4C). As shown in Figure 4D, the inhibitory phosphorylation of GSK-3β at Ser9 and Akt was reduced in SW620-NDRG2 cell line, which activated the kinase activity of GSK-3β and induced the degradation of β-catenin protein. The effect of NDRG2 on TCF/β-catenin signaling was verified by RT–PCR analysis of TCF/LEF target genes. Cyclin D1 and fibronectin are known to have TCF-/LEF-binding sites in their promoter regions and their transcription was decreased by NDRG2 expression (Figure 4E). However, their expression was not changed in SW620-NDRG2T334A and SW620-NDRG2Δ302, indicating that NDRG2 phosphorylation in its C-terminal region is critical to the regulation of the TCF/LEF transcription activity. As expected, NDRG2T334A and NDRG2Δ302 showed no effect on the phosphorylation status of GSK-3β or on the degradation of β-catenin (Figures 3D and 4D).


NDRG2 expression decreases with tumor stages and regulates TCF/beta-catenin signaling in human colon carcinoma.

Kim YJ, Yoon SY, Kim JT, Song EY, Lee HG, Son HJ, Kim SY, Cho D, Choi I, Kim JH, Kim JW - Carcinogenesis (2009)

NDRG2 regulates the phosphorylation of GSK-3β. (A) NDRG2 introduction attenuated the increase of TCF/LEF activity following LiCl or β-catenin treatment. (B) Mutant NDRG2, NDRG2T334A and NDRG2Δ302 showed no effect on the transcription activity of TCF/LEF. (C) SW620/N was treated with NDRG2 siRNA. The reduction of NDRG2 mRNA and protein levels by siRNA was confirmed by RT–PCR and western blot analyses. The protein level of β-catenin was increased in the NDRG2 siRNA-introduced cells. (D) The suppression of LiCl-induced TCF activation by NDRG2 presented the possible function of NDRG2 in the regulation of GSK-3β activity, and the inhibitory phosphorylation of GSK-3β was reduced by NDRG2 overexpression. (E) Target genes of TCF/LEF transcription factor were downregulated following NDRG2 introduction.
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Related In: Results  -  Collection

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fig4: NDRG2 regulates the phosphorylation of GSK-3β. (A) NDRG2 introduction attenuated the increase of TCF/LEF activity following LiCl or β-catenin treatment. (B) Mutant NDRG2, NDRG2T334A and NDRG2Δ302 showed no effect on the transcription activity of TCF/LEF. (C) SW620/N was treated with NDRG2 siRNA. The reduction of NDRG2 mRNA and protein levels by siRNA was confirmed by RT–PCR and western blot analyses. The protein level of β-catenin was increased in the NDRG2 siRNA-introduced cells. (D) The suppression of LiCl-induced TCF activation by NDRG2 presented the possible function of NDRG2 in the regulation of GSK-3β activity, and the inhibitory phosphorylation of GSK-3β was reduced by NDRG2 overexpression. (E) Target genes of TCF/LEF transcription factor were downregulated following NDRG2 introduction.
Mentions: Protein stability of β-catenin is known to be regulated by the kinase activity of GSK-3β that seemed to be affected by NDRG2 function. TCF/LEF activity in SW620 cells was enhanced by treatment with LiCl, a well-known GSK-3β inhibitor, as well as by cotransfection of β-catenin. NDRG2-induced decrease of TCF/LEF activity was also detected under these enhanced conditions (Figure 4A). The data that NDRG2 attenuated the increase of TCF/LEF activity following LiCl treatment support the notion that NDRG2 may regulate the kinase activity of GSK-3β. Mutants NDRG2, NDRG2T334A, NDRG2T334D and NDRG2Δ302 showed no effect on the transcription activity of TCF/LEF (Figure 4B). Otherwise, as the expression of NDRG2 was downregulated by NDRG2 siRNA, the protein level of β-catenin was increased (Figure 4C). As shown in Figure 4D, the inhibitory phosphorylation of GSK-3β at Ser9 and Akt was reduced in SW620-NDRG2 cell line, which activated the kinase activity of GSK-3β and induced the degradation of β-catenin protein. The effect of NDRG2 on TCF/β-catenin signaling was verified by RT–PCR analysis of TCF/LEF target genes. Cyclin D1 and fibronectin are known to have TCF-/LEF-binding sites in their promoter regions and their transcription was decreased by NDRG2 expression (Figure 4E). However, their expression was not changed in SW620-NDRG2T334A and SW620-NDRG2Δ302, indicating that NDRG2 phosphorylation in its C-terminal region is critical to the regulation of the TCF/LEF transcription activity. As expected, NDRG2T334A and NDRG2Δ302 showed no effect on the phosphorylation status of GSK-3β or on the degradation of β-catenin (Figures 3D and 4D).

Bottom Line: SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid.TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis.Intracellular beta-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of beta-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Republic of Korea.

ABSTRACT
NDRG (N-Myc downstream-regulated gene)-2 is a member of the NDRG family. Although it has been suggested that NDRG2 is involved in cellular differentiation and tumor suppression, its intracellular signal and regulatory mechanism are not well known. Here, we show the differential expression of NDRG2 in human colon carcinoma cell lines and tissues by reverse transcription-polymerase chain reaction and immunohistochemical analyses with monoclonal antibody against NDRG2. NDRG2 was strongly expressed in normal colonic mucosa and colonic adenomatous tissues (25 of 25) but not in all invasive cancer tissues [44 of 99 (44%)]. Most distinctive results indicated that the high expression level of NDRG2 has a positive correlation with tumor differentiation and inverse correlation with tumor invasion depth and Dukes' stage of colon adenocarcinoma. To investigate the roles of NDRG2 in tumorigenesis, we used in vitro cell culture system. SW620 colon cancer cell line with a low level of intrinsic NDRG2 protein was transfected with NDRG2-expressing plasmid. TOPflash luciferase reporter assay showed that the transcriptional activity of T-cell factor (TCF)/lymphoid enhancer factor (LEF) was reduced by NDRG2 introduction, but not by the introduction of mutant NDRG2 generated by deletion or site-directed mutagenesis. Intracellular beta-catenin levels were slightly reduced in the NDRG2-transfected SW620 cells and this regulation of beta-catenin stability and TCF/LEF activity were mediated through the modulation of glycogen synthase kinase-3beta activity by NDRG2 function. Our results suggest that NDRG2 might play a pivotal role as a potent tumor suppressor by the attenuation of TCF/beta-catenin signaling for the maintenance of healthy colon tissues.

Show MeSH
Related in: MedlinePlus