SIRT2 interacts with β-catenin to inhibit Wnt signaling output in response to radiation-induced stress

Abstract

Wnt signaling is critical to maintaining cellular homeostasis via regulation of cell division, mitigation of cell stress, and degradation. Aberrations in Wnt signaling contribute to carcinogenesis and metastasis, whereas sirtuins have purported roles in carcinogenesis, aging, and neurodegeneration. Therefore, the hypothesis that sirtuin 2 (SIRT2) directly interacts with β-catenin and whether this interaction alters the expression of Wnt target genes to produce an altered cellular phenotype was tested. Coimmunoprecipitation studies, using mouse embryonic fibroblasts (MEF) from Sirt2 wild-type and genomic knockout mice, demonstrate that β-catenin directly binds SIRT2. Moreover, this interaction increases in response to oxidative stress induced by ionizing radiation. In addition, this association inhibits the expression of important Wnt target genes such as survivin (BIRC5), cyclin D1 (CCND1), and c-myc (MYC). In Sirt2 null MEFs, an upregulation of matrix metalloproteinase 9 (MMP9) and decreased E-cadherin (CDH1) expression is observed that produces increased cellular migration and invasion. Together, these data demonstrate that SIRT2, a tumor suppressor lost in multiple cancers, inhibits the Wnt signaling pathway in nonmalignant cells by binding to β-catenin and that SIRT2 plays a critical role in the response to oxidative stress from radiation.

Implications: Disruption of the SIRT2-β-catenin interaction represents an endogenous therapeutic target to prevent transformation and preserve the integrity of aging cells against exogenous stressors such as reactive oxygen species.

Figure 1

Nuclear β-catenin levels are elevated in Sirt2 KO MEFs. A, immunoblot analysis of enriched nuclear and cytoplasmic fractions for β-catenin, lamin B (nuclear marker) or manganese superoxide dismutase (cytoplasmic marker) showing increased nuclear β-catenin protein level in Sirt2 KO MEFs. B, densitometric analysis of the subcellular fractionation. ***, P < 0.001. C, immunoblot analysis showing increased pY142 β-catenin in Sirt2 KO MEFs. D, β-catenin immunofluoresescent staining showing markedly increased level of nuclear β-catenin in Sirt2 KO MEFs vs. WT.

Figure 2

SIRT2 interacts directly with β-catenin. A, coimmunoprecipitation showing association of SIRT2 and β-catenin increases with IR. Sirt2 WT MEFs were exposed to single fractions of 2Gy, 5 Gy and 10 Gy, and compared to sham controls. Cells were harvested 24h after radiation and immunoprecipitated either with normal rabbit IgG control, SIRT2 or β-catenin antibodies. Bound proteins were analyzed by immunoblot with β-catenin antibody. B, coimmunoprecipitation analysis showing SIRT2 associates with β-catenin in PC-3 cells. C, coimmunoprecipitation analysis showing overexpressed Flag-SIRT2 binds to β-catenin. Protein lysates from U87 glioma cells stably expressing either pcDNA3 or Flag-SIRT2 were subjected to immunoprecipitation using rabbit IgG, DDK or β-catenin antibodies. Bound proteins were analyzed by immunoblot with β-catenin antibody. D, ROS measurement. Sirt2 WT and KO MEFs were labeled with H₂DCFDA or carboxy DCFDA for 30 min before exposure to a single fraction of 10 Gy compared to sham controls. Cells were harvested after 30 min and analyzed. Treatment with PEG-catalase was used to scavenge ROS generated by radiation. E, Clonogenic survival assay. Sirt2 WT and KO cells were plated for clonogenic survival assay and treated with 200U/ml PEG-cat for 2h, followed by radiation. PEG-cat was left for the duration of the assay. Values represent the mean +/− SEM. *, P < 0.05, ***, P < 0.005

Figure 3

Acetylation of β-catenin and activation of the Wnt signaling pathway in Sirt2 KO MEFs. A, acetylated β-catenin immunofluorescent staining showing markedly increased level of acetylated β-catenin present in Sirt2 KO MEFs compared to WT. B, increased acetylated β-catenin is present in Sirt2 KO MEFs. Whole cell lysates from Sirt2 WT and KO cells were immunoprecipitated with rabbit IgG control or β-catenin antibody. Pulled-down complexes were immunoblotted with acetylated lysine or acetylated β-catenin antibody. C, SIRT2 deacetylates β-catenin in vitro. Immunoprecipitated β-catenin from Sirt2 KO MEFs was incubated with or without purified recombinant Sirt2 in the presence or absence of NAD+. The reaction mixtures were separated by SDS-PAGE and immunoblotted with antibodies against acetylated lysine, β-catenin or SIRT2. D, immunoblot showing upregulated level of activated, phosphorylated GSK-3-β in Sirt2 KO MEFs compared to WT.

Figure 4

SIRT2 inhibits Wnt target gene expression. A, real-time PCR showing upregulated mRNA levels of cyclin D1, c-myc and survivin in Sirt2 KO MEFs compared with WT. **, P < 0.005, ***, P < 0.0005. B, immunoblot showing increased protein levels of c-myc, cyclin D1, survivin and c-Jun in Sirt2 KO compared to WT. C, real-time PCR showing that knockdown of Sirt2 upregulates cyclin D1, c-myc and survivin mRNA levels. ***, P < 0.001. D, immunoblot showing knockdown of Sirt2 upregulates c-myc, cyclin D1 and survivin protein expression.

Figure 5

SIRT2 inhibits Wnt Signaling. A, pTOPFLASH luciferase promoter analysis showing upregulated TCF/LEF transcriptional activity in Sirt2 KO MEFs compared to WT. **, P < 0.005. B, ChIP analysis showing increased binding of β-catenin to the promoters of c-myc, cyclin D1, and survivin. *, P < 0.5, **, P < 0.01.

Figure 6

SIRT2 inhibits cell motility/invasion and MMP9 expression and upregulates E-cadherin expression. A, cell motility analysis showing Sirt2 KO MEFs migrated faster through uncoated Boyden chambers compared to WT in response to serum. B, cell invasion analysis showing Sirt2 KO fibroblasts migrated faster through collagen-coated Boyden chambers in response to serum. ***, P < 0.0001. C, cell invasion analysis showing that knockdown of Sirt2 slowed the migration of Sirt2 WT MEFs through collagen-coated Boyden chambers. **, P,< 0.005. D, real-time PCR showing upregulated Mmp9 mRNA in Sirt2 KO MEFs compared to WT. **, P < 0.005. E, immunoblot showing increased MMP9 in Sirt2 KO cells compared to WT. F, immunoblot showing markedly decreased E-cadherin in Sirt2 KO cells compared to WT. Data from A–C in this figure are representative of one of three independent experiments. Data from D–F in this figure show means ± SEM from three independent experiments.