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OGA heterozygosity suppresses intestinal tumorigenesis in Apc(min/+) mice.

Yang YR, Jang HJ, Yoon S, Lee YH, Nam D, Kim IS, Lee H, Kim H, Choi JH, Kang BH, Ryu SH, Suh PG - Oncogenesis (2014)

Bottom Line: In two independent microarray data sets, the expression of OGA and OGT was significantly associated with poor cancer-specific survival of colorectal cancer (CRC) patients.Apc(min/+) OGA(+/-) mice exhibited a significantly increased survival rate compared with Apc(min/+) mice.However, the knockout of OGA did not affect Wnt/β-catenin signaling.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.

ABSTRACT
Emerging evidence suggests that aberrant O-GlcNAcylation is associated with tumorigenesis. Many oncogenic factors are O-GlcNAcylated, which modulates their functions. However, it remains unclear how O-GlcNAcylation and O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), affect the development of cancer in animal models. In this study, we show that reduced level of OGA attenuates colorectal tumorigenesis induced by Adenomatous polyposis coli (Apc) mutation. The levels of O-GlcNAcylation and O-GlcNAc cycling enzymes were simultaneously upregulated in intestinal adenomas from mice, and in human patients. In two independent microarray data sets, the expression of OGA and OGT was significantly associated with poor cancer-specific survival of colorectal cancer (CRC) patients. In addition, OGA heterozygosity, which results in increased levels of O-GlcNAcylation, attenuated intestinal tumor formation in the Apc(min/+) background. Apc(min/+) OGA(+/-) mice exhibited a significantly increased survival rate compared with Apc(min/+) mice. Consistent with this, Apc(min/+) OGA(+/-) mice expressed lower levels of Wnt target genes than Apc(min/+). However, the knockout of OGA did not affect Wnt/β-catenin signaling. Overall, these findings suggest that OGA is crucial for tumor growth in CRC independently of Wnt/β-catenin signaling.

No MeSH data available.


Related in: MedlinePlus

Elevated O-GlcNAcylation does not affect Wnt/β-catenin signaling. (a) β-Catenin is O-GlcNAcylated. HEK-293 cells were transfected with empty vector or a GFP-β-catenin expression vector. GFP-β-catenin-transfected HEK-293 cells were untreated (−) or treated (+) with Thiamet G. Cell lysates were immunoprecipitated with anti-GFP antibodies and immunoblotting was performed using antibodies against O-GlcNAc (RL-2). (b) Wild-type and OGA+/− MEFs were stimulated with Wnt3a (100 ng/ml) at the indicated time points, and then subjected to immunoblotting. (c) Axin2 and Jun mRNA levels were analyzed by qPCR after Wnt3a treatment for 6 h. (d) Control, OGA knockdown and Thiamet G (#SML0244; Sigma, Madison, WI, USA)-treated HEK-293 cells were stimulated with Wnt3a-conditioned media (CM), and analyzed by western blotting at the indicated time points. (e) The same samples were analyzed by qPCR after 6 h of stimulation. Wnt3a-conditioned media were prepared from mouse L cells (ATCC, Manassas, VA, USA) stably expressing Wnt3a. The primer sequences (human) used were: Axin2 forward, 5′-ATGAGTAGCGCCGTGTTAGTG-3′ Axin2 reverse, 5′-GGGCATAGGTTTGGTGGACT-3′ GAPDH forward, 5′-CCACTCCTCCACCTTTGAC-3′ and GAPDH reverse, 5′-ACCCTGTTGCTGTAGCCA-3′.
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fig4: Elevated O-GlcNAcylation does not affect Wnt/β-catenin signaling. (a) β-Catenin is O-GlcNAcylated. HEK-293 cells were transfected with empty vector or a GFP-β-catenin expression vector. GFP-β-catenin-transfected HEK-293 cells were untreated (−) or treated (+) with Thiamet G. Cell lysates were immunoprecipitated with anti-GFP antibodies and immunoblotting was performed using antibodies against O-GlcNAc (RL-2). (b) Wild-type and OGA+/− MEFs were stimulated with Wnt3a (100 ng/ml) at the indicated time points, and then subjected to immunoblotting. (c) Axin2 and Jun mRNA levels were analyzed by qPCR after Wnt3a treatment for 6 h. (d) Control, OGA knockdown and Thiamet G (#SML0244; Sigma, Madison, WI, USA)-treated HEK-293 cells were stimulated with Wnt3a-conditioned media (CM), and analyzed by western blotting at the indicated time points. (e) The same samples were analyzed by qPCR after 6 h of stimulation. Wnt3a-conditioned media were prepared from mouse L cells (ATCC, Manassas, VA, USA) stably expressing Wnt3a. The primer sequences (human) used were: Axin2 forward, 5′-ATGAGTAGCGCCGTGTTAGTG-3′ Axin2 reverse, 5′-GGGCATAGGTTTGGTGGACT-3′ GAPDH forward, 5′-CCACTCCTCCACCTTTGAC-3′ and GAPDH reverse, 5′-ACCCTGTTGCTGTAGCCA-3′.

Mentions: The activation of β-catenin is essential not only for tumor initiation but also for tumor progression in Apcmin/+ mice.21 Importantly, β-catenin is directly O-GlcNAcylated, and we also observed that β-catenin is modified with O-GlcNAc (Figure 4a). The O-GlcNAcylation of β-catenin regulates its localization and transcriptional activity.22 OGT interacts with β-catenin to regulate cyclin D1 synthesis upon serum stimulation.12 Therefore, we assessed whether OGA deficiency affects Wnt/β-catenin signaling. The deletion of OGA did not affect Wnt3-mediated β-catenin accumulation (Figure 4b). In addition, there were no significant differences in Axin2 and Jun mRNA levels between OGA+/+ and OGA−/− mouse embryonic fibroblasts after stimulation with Wnt3a (Figure 4c). We also used immunoblotting and quantitative PCR to further confirm that the OGA inhibitor Thiamet G and knockdown did not affect Wnt/β-catenin signaling (Figures 4d and e). Increased O-GlcNAcylation after Thiamet G treatment might result in the upregulation OGA to compensate for the increased O-GlcNAcylation. This observation supports the results presented in Figure 3a. These results suggest that OGA heterozygosity affects tumorigenesis independently of Wnt/β-catenin signaling. In addition, intestinal tumorigenesis correlates with OGT and OGA levels, which are important for the dynamic regulation of O-GlcNAcylation.


OGA heterozygosity suppresses intestinal tumorigenesis in Apc(min/+) mice.

Yang YR, Jang HJ, Yoon S, Lee YH, Nam D, Kim IS, Lee H, Kim H, Choi JH, Kang BH, Ryu SH, Suh PG - Oncogenesis (2014)

Elevated O-GlcNAcylation does not affect Wnt/β-catenin signaling. (a) β-Catenin is O-GlcNAcylated. HEK-293 cells were transfected with empty vector or a GFP-β-catenin expression vector. GFP-β-catenin-transfected HEK-293 cells were untreated (−) or treated (+) with Thiamet G. Cell lysates were immunoprecipitated with anti-GFP antibodies and immunoblotting was performed using antibodies against O-GlcNAc (RL-2). (b) Wild-type and OGA+/− MEFs were stimulated with Wnt3a (100 ng/ml) at the indicated time points, and then subjected to immunoblotting. (c) Axin2 and Jun mRNA levels were analyzed by qPCR after Wnt3a treatment for 6 h. (d) Control, OGA knockdown and Thiamet G (#SML0244; Sigma, Madison, WI, USA)-treated HEK-293 cells were stimulated with Wnt3a-conditioned media (CM), and analyzed by western blotting at the indicated time points. (e) The same samples were analyzed by qPCR after 6 h of stimulation. Wnt3a-conditioned media were prepared from mouse L cells (ATCC, Manassas, VA, USA) stably expressing Wnt3a. The primer sequences (human) used were: Axin2 forward, 5′-ATGAGTAGCGCCGTGTTAGTG-3′ Axin2 reverse, 5′-GGGCATAGGTTTGGTGGACT-3′ GAPDH forward, 5′-CCACTCCTCCACCTTTGAC-3′ and GAPDH reverse, 5′-ACCCTGTTGCTGTAGCCA-3′.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig4: Elevated O-GlcNAcylation does not affect Wnt/β-catenin signaling. (a) β-Catenin is O-GlcNAcylated. HEK-293 cells were transfected with empty vector or a GFP-β-catenin expression vector. GFP-β-catenin-transfected HEK-293 cells were untreated (−) or treated (+) with Thiamet G. Cell lysates were immunoprecipitated with anti-GFP antibodies and immunoblotting was performed using antibodies against O-GlcNAc (RL-2). (b) Wild-type and OGA+/− MEFs were stimulated with Wnt3a (100 ng/ml) at the indicated time points, and then subjected to immunoblotting. (c) Axin2 and Jun mRNA levels were analyzed by qPCR after Wnt3a treatment for 6 h. (d) Control, OGA knockdown and Thiamet G (#SML0244; Sigma, Madison, WI, USA)-treated HEK-293 cells were stimulated with Wnt3a-conditioned media (CM), and analyzed by western blotting at the indicated time points. (e) The same samples were analyzed by qPCR after 6 h of stimulation. Wnt3a-conditioned media were prepared from mouse L cells (ATCC, Manassas, VA, USA) stably expressing Wnt3a. The primer sequences (human) used were: Axin2 forward, 5′-ATGAGTAGCGCCGTGTTAGTG-3′ Axin2 reverse, 5′-GGGCATAGGTTTGGTGGACT-3′ GAPDH forward, 5′-CCACTCCTCCACCTTTGAC-3′ and GAPDH reverse, 5′-ACCCTGTTGCTGTAGCCA-3′.
Mentions: The activation of β-catenin is essential not only for tumor initiation but also for tumor progression in Apcmin/+ mice.21 Importantly, β-catenin is directly O-GlcNAcylated, and we also observed that β-catenin is modified with O-GlcNAc (Figure 4a). The O-GlcNAcylation of β-catenin regulates its localization and transcriptional activity.22 OGT interacts with β-catenin to regulate cyclin D1 synthesis upon serum stimulation.12 Therefore, we assessed whether OGA deficiency affects Wnt/β-catenin signaling. The deletion of OGA did not affect Wnt3-mediated β-catenin accumulation (Figure 4b). In addition, there were no significant differences in Axin2 and Jun mRNA levels between OGA+/+ and OGA−/− mouse embryonic fibroblasts after stimulation with Wnt3a (Figure 4c). We also used immunoblotting and quantitative PCR to further confirm that the OGA inhibitor Thiamet G and knockdown did not affect Wnt/β-catenin signaling (Figures 4d and e). Increased O-GlcNAcylation after Thiamet G treatment might result in the upregulation OGA to compensate for the increased O-GlcNAcylation. This observation supports the results presented in Figure 3a. These results suggest that OGA heterozygosity affects tumorigenesis independently of Wnt/β-catenin signaling. In addition, intestinal tumorigenesis correlates with OGT and OGA levels, which are important for the dynamic regulation of O-GlcNAcylation.

Bottom Line: In two independent microarray data sets, the expression of OGA and OGT was significantly associated with poor cancer-specific survival of colorectal cancer (CRC) patients.Apc(min/+) OGA(+/-) mice exhibited a significantly increased survival rate compared with Apc(min/+) mice.However, the knockout of OGA did not affect Wnt/β-catenin signaling.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea.

ABSTRACT
Emerging evidence suggests that aberrant O-GlcNAcylation is associated with tumorigenesis. Many oncogenic factors are O-GlcNAcylated, which modulates their functions. However, it remains unclear how O-GlcNAcylation and O-GlcNAc cycling enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), affect the development of cancer in animal models. In this study, we show that reduced level of OGA attenuates colorectal tumorigenesis induced by Adenomatous polyposis coli (Apc) mutation. The levels of O-GlcNAcylation and O-GlcNAc cycling enzymes were simultaneously upregulated in intestinal adenomas from mice, and in human patients. In two independent microarray data sets, the expression of OGA and OGT was significantly associated with poor cancer-specific survival of colorectal cancer (CRC) patients. In addition, OGA heterozygosity, which results in increased levels of O-GlcNAcylation, attenuated intestinal tumor formation in the Apc(min/+) background. Apc(min/+) OGA(+/-) mice exhibited a significantly increased survival rate compared with Apc(min/+) mice. Consistent with this, Apc(min/+) OGA(+/-) mice expressed lower levels of Wnt target genes than Apc(min/+). However, the knockout of OGA did not affect Wnt/β-catenin signaling. Overall, these findings suggest that OGA is crucial for tumor growth in CRC independently of Wnt/β-catenin signaling.

No MeSH data available.


Related in: MedlinePlus