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. 2014 Nov;21(11):1769-79.
doi: 10.1038/cdd.2014.88. Epub 2014 Jun 27.

Ablation of galectin-3 Induces p27(KIP1)-dependent Premature Senescence Without Oncogenic Stress

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Free PMC article

Ablation of galectin-3 Induces p27(KIP1)-dependent Premature Senescence Without Oncogenic Stress

S-J Kim et al. Cell Death Differ. .
Free PMC article

Abstract

Premature senescence induced by oncogenic stimuli or tumor suppressor activation plays opposing roles in tumorigenesis. Here, we propose that galectin-3, a β-galactoside-binding lectin, regulates premature senescence without oncogenic stress. We detected premature senescence, decreased Skp2, and increased p27(KIP1) expression in galectin-3 knockout MEFs and galectin-3-depleted gastric cancer cells. Interestingly, galectin-3 depletion did not affect other senescence inducers such as p14(ARF), p16(INK4A), and p21(WAF1/CIP1), suggesting that galectin-3-regulated senescence is p27(KIP1) dependent. We demonstrate that galectin-3 depletion decreases retinoblastoma protein (Rb) phosphorylation (Ser780, Ser807/811), cyclin D1 and CDK4 expression, and E2F1 transcriptional activation. Galectin-3 directly interacts with the cyclin D1/CDK4 complex and promotes hyperphosphorylation of Rb. It also blocks the inhibition of E2F1 transcription, thereby increasing the expression of Skp2 and reducing the stability of p27(KIP1) to promote the proliferation of gastric cancer cells. Xenograft mice with galectin-3-depleted gastric cancer cells display tumor growth retardation that is reversed by Skp2 overexpression. Increased expression of galectin-3 is also associated with the advanced TNM (tumor, lymph node, metastasis) system, clinicopathological stage, and lymph node metastases. The probability of survival was significantly decreased in gastric cancer patients with galectin-3(high) p27(KIP1-low)cells. Taken together, our results show that galectin-3 may accelerate gastric tumorigenesis by inhibiting premature senescence.

Figures

Figure 1
Figure 1
Inhibition of galectin-3 reduces the proliferation of gastric cancer cells in a Skp2- and p27KIP1-dependent manner. (a) Cell proliferation after silencing of galectin-3 using siRNA transfection with respect to time (1–3 days) and concentration (1–20 nM) in the AGS human gastric cancer cells. The error bars indicate 95% confidence intervals; *, **P<0.0001 using one-way ANOVA. (b) Detection of the levels of galectin-3, Skp2, p27KIP1, p14ARF, p16INK4A, and p21WAF1/CIP1 after silencing of galectin-3 using siRNA transfection in the AGS human gastric cancer cells. β-Actin was used as the normalization control. (c) Detection of mRNA and protein expression of galectin-3, p27KIP1, and Skp2 after transfection with galectin-3 siRNA for 48 h, as analyzed by RT-PCR and immunoblotting, respectively. β-Actin was used as the normalization control. (d and e) After transfection of AGS cells with galectin-3 siRNA, p27KIP1 siRNA, and co-transfection of AGS cells with both siRNAs (d) the protein expression of galectin-3, p27KIP1, and Skp2 was analyzed by western blotting. β-Actin was used as the normalization control. (e) Cell cycle populations were examined by PI staining. A quantitative graph (left panel) and histogram (right panel) are generated. Marking of histogram is as follows: M1-SubG1, M2-G1, M3-S, and M4-G2
Figure 2
Figure 2
Decreased cell proliferation and increased premature senescence in galectin-3 knockout mouse embryonic fibroblasts (MEFs) and galectin-3-depleted human skin fibroblasts. (a) Expression levels of galectin-3 in galectin-3−/− and galectin-3+/+ MEFs were analyzed by western blotting (upper panel). Galectin-3−/− and galectin-3+/+ MEFs were stained with crystal violet (lower panel), and the graph shows the percentage of cells in the microscopic fields (right panel). The error bars indicate 95% confidence intervals; *P=0.0397 using the two-sided t-test. (b) Cell lysates of galectin-3−/− and galectin-3+/+ MEFs were prepared and analyzed by RT-PCR using primers specific for p27KIP1, Skp2, and galectin-3. GAPDH served as the normalization control. Protein levels were also detected using antibodies against p27KIP1, p21WAF1/CIP1, Skp2, and galectin-3 by western blotting. β-Actin was used as the normalization control. (c) Premature senescence was induced in galectin-3−/− MEFs. Galectin-3−/− and galectin-3+/+ MEFs were stained for β-galactosidase activity. The graph shows the percentage of β-galactosidase-positive cells (right panel). The error bars indicate 95% confidence intervals; *P<0.0001 using the two-sided t-test. The scale bar indicates 100 μm. (d) After 48 h of transfection with control siRNA, galectin-3 siRNA, p27KIP1 siRNA, and both siRNAs in human skin fibroblasts, the cells were stained with crystal violet. (e) Cell lysates were subjected to western blotting using the indicated antibodies. (f) Premature senescence was detected by β-galactosidase activity. The graph shows the percentage of β-galactosidase-positive cells (right panel). The error bars indicate 95% confidence intervals; *P<0.0001 and **P=0.0003 using the two-sided t-test
Figure 3
Figure 3
Increased premature senescence caused by the inhibition of galectin-3 is p27KIP1 dependent, but p53 independent. (a) After 48 h of transfection with control siRNA, galectin-3 siRNA, p27KIP1siRNA, and both siRNAs in the (a and b) AGS cells and (c and d) SNU601 cells, (a) cell proliferation was evaluated. The error bars indicates 95% confidence intervals; * P<0.0001 and **P=0.0047 using a two-sided t-test. (b) Premature senescence was detected by β-galactosidase activity. The graph shows the percentage of β-galactosidase-positive cells. The error bars indicate 95% confidence intervals; *P<0.0001 and **P<0.0001 using the two-sided t-test. (c) Cell proliferation was determined and the data were presented as a histogram. The error bars indicate 95% confidence intervals; *P<0.0001 and **P=0.0139 using the two-sided t-test. (d) Premature senescence was analyzed by determining the β-galactosidase activity. The graph shows the percentage of β-galactosidase-positive cells. The error bars indicate 95% confidence intervals; *P=0.0008 and **P=0.0013 using the two-sided t-test. (eg) After transfection with the galectin-3 plasmids, Skp2 siRNA was additionally transfected into the SNU-638 cells. (e) Expression of the indicated proteins was detected by western blotting. (f) Cell proliferation and (g) cell cycle population analyses were performed by Ez-Cytox assay and PI staining, respectively. The error bars indicate 95% confidence intervals; *P=0.0005 and **P=0.0067 using the two-sided t-test
Figure 4
Figure 4
Inhibition of Skp2 reduces cell proliferation and induces premature senescence through an increase of p27KIP1 protein expression. (ac) After transfection with the Skp2 plasmids, galectin-3 siRNA was additionally transfected into the AGS cells. (a) Protein expression, (b) cell proliferation, and (c) cell cycle population analyses were performed by western blotting, Ez-Cytox assay, and PI staining, respectively. The error bars indicate 95% confidence intervals; *P=0.0094 using the two-sided t-test. AGS cells were transfected with control siRNA, Skp2 siRNA, p27KIP1siRNA, or both siRNAs for 48 h; (d) the protein expression was analyzed by western blotting and (e) cell proliferation was evaluated and the data are presented as a histogram. The error bars indicate 95% confidence intervals; *P=0.0005 and **P=0.0156 using the two-sided t-test. (f) Premature senescence was detected by β-galactosidase activity. The graph shows the percentage of β-galactosidase-positive cells. The error bars indicate 95% confidence intervals; *P=0.0013 and **P=0.0017 using the two-sided t-test. (g) The cell cycle populations were analyzed by PI staining
Figure 5
Figure 5
Inhibition or overexpression of galectin-3 regulates the phosphorylation of Rb and expression levels of cyclin D1 and CDK4. (a) Detection of protein levels of ppRb (at Ser 780 and Ser 807/811), Rb, E2F1, cyclin D1, and CDK4 after the depletion of galectin-3 using siRNA transfection in the AGS human gastric cancer cells. (b) Detection of the protein levels of galectin-3, Skp2, Rb, ppRb (at Ser 780 and Ser 807/811), and p27KIP1 by western blot analysis in SNU-638 cells infected with a lentiviral construct containing galectin-3 or LacZ as the negative control. (c) Detection of the levels of galectin-3, Skp2, Rb, ppRb (at Ser 780 and Ser 807/811), and p27KIP1 by western blot analysis after transfection of cyclin D1 and CDK4 in SNU-638 cells infected with the constructs in (b). (d) Schematic model of Flag-galectin-3 domain (1–250 aa as the full length; 1–110 aa as the N-terminal tail; and 33–250, 63–250, and 111–250 aa as the CRD). (e) Detection of the levels of galectin-3, Skp2, Rb, ppRb (at Ser 780 and Ser 807/811), and p27KIP1 by western blot analysis after transfection of the galectin-3 domains in SNU-638 cells. β-Actin was used as the normalization control
Figure 6
Figure 6
Galectin-3 directly interacts with Rb and regulates the transcriptional activity of E2F1. (a) Immunoprecipitation was performed with antibodies against galectin-3 and Rb to detect the interactions of galectin-3 with E2F1, Rb, cyclin D1, and CDK4 in the AGS cells. Whole cell lysate was used as the positive control. (b) Immunoprecipitation with Flag-galectin-3 domains, Cyclin D1, CDK4, and Rb using transfection of the galectin-3 domains in SNU-638 cells. (c) Schematic model of the interaction of the Skp2 promoter with the E2F1 binding site. ChIP primers were prepared to detect the E2F1 binding site (−42 to −35) from −95 to +135. The ChIP assay was performed using antibodies against galectin-3, Rb, and E2F1 in scRNA- and galectin-3 siRNA-transfected AGS cells. A PCR primer for the Skp2 promoter was used to detect promoter fragments in the immunoprecipitates. The input lane with total genomic DNA was used as a control for the PCR reaction. (d–f) Detection of luciferase activity after transfection of (d) SNU638 cells with an E2F1 consensus plasmid and galectin-3 domains for 48 h, (e) AGS cells with siRNA targeting galectin-3, Cyclin D1, and CDK4, or LacZ as a negative control for 48 h, and (f) galectin-3-overexpressing SNU638 cells with siRNA targeting Cyclin D1 and CDK4 for 48 h. The cells were transfected with 1 μg of the E2F1 consensus plasmid. The error bars indicate 95% confidence intervals; *P<0.0001 using a two-sided t-test. Three independent experiments were performed
Figure 7
Figure 7
Galectin-3 depletion reduces the tumor burden in gastric cancer cell-xenografted mice and these effects are reversed by overexpression of skp2. (a–d) Lentiviruses expressing galectin-3 shRNA and overexpressing Skp2 were employed to produce stable AGS cell lines. Lentivirus expressing shRNA targeting LacZ was used as a control. Mice (n=5 per group) were inoculated subcutaneously into both flanks with 106 cells of each of the AGS cell lines. (a) Skp2 overexpression and galectin-3 depletion were confirmed by western blot analysis. (b) Tumor formation was observed 30 days after inoculation. (c) Tumor formation was quantified by measuring the tumor volume 10 days after inoculation (n=5). The error bars indicate 95% confidence intervals; *P=0.001 and **P=0.0015 using two-sided t-test. All statistical tests were two sided. (d) Schematic model of galectin-3-dependent promotion of tumor progression and premature senescence. Galectin-3 interacts with Cyclin D1, CDK4, and Rb and blocks the inhibition of E2F1 transcription. This increases the expression of Skp2 and reduces the stability of p27KIP1 to promote the proliferation of gastric cancer cells. After galectin-3 depletion, Rb interacts with E2F1 to block the transcriptional activity of E2F1, suppress Skp2 expression, and increase the stability of p27KIP1. This results in the suppression of the proliferation of gastric cancer cells and in the induction of premature cellular senescence
Figure 8
Figure 8
Positive association between galectin-3 and Skp2 expression and negative association between galectin-3 and p27KIP1 expression is found in malignant tissues of gastric cancer patients. (a) The correlation between the mRNA expression of galectin-3 and Skp2 in the malignant tissues of 52 gastric cancer patients. The mRNA expression was detected by RT-PCR and quantified by an NIH ImageJ analyzer. GAPDH and β-actin were used as normalization controls. (b) The probability of survival of gastric cancer patients whose tumors show high galectin-3 and low p27KIP1 levels is (red line) compared with that of gastric cancer patients whose tumors show low galectin-3 and high p27KIP1 levels (blue line) using Kaplan–Meyer analysis. Statistical analysis is described in the Materials and Methods section. (c) Protein expression of galectin-3, Skp2, and p27KIP1 in the malignant tissues of gastric cancer patients, as shown by immunohistochemical staining (brown) with hematoxylin and eosin (H&E). The stained tissue samples were observed using an inverted light microscope. Magnification: × 200 (top) and × 400 (bottom)

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