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. 2016 Aug 30;7(35):57117-57130.
doi: 10.18632/oncotarget.10986.

Ablation of Human Telomerase Reverse Transcriptase (hTERT) Induces Cellular Senescence in Gastric Cancer Through a galectin-3 Dependent Mechanism

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Free PMC article

Ablation of Human Telomerase Reverse Transcriptase (hTERT) Induces Cellular Senescence in Gastric Cancer Through a galectin-3 Dependent Mechanism

Sun-Hyuk La et al. Oncotarget. .
Free PMC article

Abstract

The human Telomerase Reverse Transcriptase (hTERT) gene encodes a rate-limiting catalytic subunit of telomerase that maintains genomic integrity. Suppression of hTERT expression could induce cellular senescence and is considered a potent approach for gastric cancer therapy. However, control of hTERT expression and function remains poorly understood in gastric cancer. In this study, we demonstrated that high expression levels of hTERT in malignant tissues are correlated with poor survival probability in gastric cancer patients. Knockdown of hTERT expression retarded cell proliferation and cellular senescence, which was confirmed by increased protein expression levels of p21cip1 and p27kip1, and decreased phosphorylation of Rb. In contrast, overexpression of hTERT increased cell proliferation and decreased cellular senescence. Remarkably, the down-regulation of hTERT expression was detected in lgals3-/- mouse embryo fibroblasts (MEFs). Knockdown of galectin-3 decreased the expression of hTERT in gastric cancer cells. Galectin-3 ablation-induced cellular senescence was rescued by concomitant overexpression of hTERT. hTERT ablation-induced cellular senescence and p21cip1 and p27kip1 expression was rescued by concomitant overexpression of galectin-3. The size of tumor burdens was increased in hTERT-overexpressed gastric cancer cells xenografted mice, whereas it was repressed by concomitant depletion of galectin-3. Additionally, we determined that the N-terminal domain of galectin-3 directly interacted with hTERT. The telomeric activity of hTERT was also decreased by galectin-3 ablation. Taken together, ablation of hTERT induces cellular senescence and inhibits the growth of gastric cancer cells, suggesting that it could be a potent target in gastric cancer therapy. We also propose that galectin-3 is an important regulator of hTERT expression and telomeric activity in gastric tumorigenesis.

Keywords: cellular senescence; galectin-3; gastric cancer; hTERT; telomerase.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Correlation between tumorigenesis and hTERT expression in gastric cancer patients
A. mRNA expression levels of hTERT of malignant tissues and normal tissues from gastric cancer patients are presented as a scatter diagram (GSE13861, GSE63089, GSE30727, GSE56807, GSE13195-2845830, 2845831, 2845837, 2845839). B. Kaplan–Meier survival plots demonstrating the poor prognostic effect due to the hTERT up-regulation, which correlated with a worse Overall Survival in gastric cancer patients (probe1:207199_at_n = 876, probe2:1555271_a_at_n = 631) (http://kmplot.com/analysis).
Figure 2
Figure 2. Ablation of hTERT altered the cell proliferation and cellular senescence of gastric cancer cells
Both YCC-2 and SNU-216 gastric cancer cells were transfected with scrambled siRNA (scRNA) and hTERT specific siRNA for 48 h. A. Cellular senescence was detected by SA-β-galactosidase activity in YCC-2 and SNU-216 gastric cancer cells. The graph shows the percentage of SA-β-galactosidase-positive cells. (P < 0.0005) B. Cell proliferation was measured by WST assay in YCC-2 and SNU-216 gastric cancer cells. (P < 0.0001) C. Detection of mRNA expression levels of hTERT, p21cip1, p27kip1, and p53 were detected by RT-PCR analysis and D. protein expression levels of these were detected by western blot analysis in YCC-2 and SNU-216 gastric cancer cells after transfection with scrambled siRNA (scRNA) and hTERT specific siRNA. β-actin was used loading control.
Figure 3
Figure 3. Overexpression of hTERT altered the cell proliferation and cellular senescence of gastric cancer cells
Both MKN-28 and SNU-638 cells were transfected with pcDNA3.0 empty vector (pcDNA3.0) and pcDNA3.0_hTERT for 48 h. A. Cellular senescence was detected by β-galactosidase activity in MKN-28 and SNU-638 gastric cancer cells after transfection with a pcDNA3.0 empty vector (pcDNA3.0) or pcDNA3.0_hTERT. The graph shows the percentage of β-galactosidase-positive cells. B. Cell proliferation was examined in MKN-28 and SNU-638 gastric cancer cell lines after transfection with pcDNA3.0 and pcDNA3.0_hTERT (P < 0.0001). C. Detection of mRNA expression levels of hTERT, p21cip1, p27kip1, and p53 were detected by RT-PCR analysis and D. protein expression levels of these were detected by western blot analysis in MKN-28 and SNU-638 cells after transfection with pcDNA3.0 and pcDNA3.0 hTERT. β-actin was used loading control.
Figure 4
Figure 4. Detection of expression level of mTERT in galectin-3 knockout mouse embryo fibroblasts (lgals3−/− KO MEFs) and expression level of galectin-3 in mTERT knockout mouse embryo fibroblasts (TERT KO MEFs)
A. mRNA expression levels of mTERT and Galectin-3 in lgals3−/− KO MEFs and B. mRNA expression levels of mTERT and galectin-3 in TERT KO MEFs were detected by RT-PCR analysis. β-actin was used loading control. C-E. SNU-638 gastric cancer cells were transfected with pcDNA3.0 alone, pcDNA3.0-galectin-3 alone, hTERT siRNA alone, and hTERT siRNA and pcDNA3.0-galectin-3 for 48 h. C. Each cell plate was stained with Crystal violet solution and showed by photographs (left panel), and cell proliferation in each cell plate was measured by WST assay and presented by quantitative graphs (right panel). D. Detection of mRNA expression levels of galectin-3, hTERT, p21cip1, p27kip1, and p53 were detected by RT-PCR analysis and protein expression levels of these were detected by western blot analysis in SNU-638 gastric cancer cells. β-actin was used loading control. E. Cellular senescence was detected by SA-β-galactosidase activity in SNU-638 cells. The graph shows the percentage of SA-β-galactosidase-positive cells.
Figure 5
Figure 5. Depletion of galectin-3 after hTERT overexpression on the cell proliferation and cellular senescence of human gastric cancer cells
A. YCC-2 gastric cancer cells were transfected with scRNA alone, pcDNA3.0-hTERT alone, pcDNA3.0-hTERT and galectin-3 siRNA, and galecitn-3 siRNA alone for 48 hr. Each cell plate was stained with Crystal violet solution and showed by photographs (left panel), and cell proliferation in each cell plate was measured by WST assay and presented by quantitative graphs (right panel). B. SNU-216 gastric cancer cells were transfected with scRNA alone, pcDNA3.0-hTERT alone, pcDNA3.0-hTERT and galectin-3 siRNA, and galecitn-3 siRNA alone for 48 hr. Each cell plate was stained with Crystal violet solution and showed by photographs (left panel), and cell proliferation in each cell plate was measured by WST assay and presented by quantitative graphs (right panel). C. Detection of mRNA expression levels of hTERT, galectin-3, p21cip1, p27kip1, and p53 were detected by RT-PCR analysis in both YCC-2 and SNU-216 gastric cancer cells. β-actin was used loading control. D. Detection of protein expression levels of hTERT, galectin-3, p21cip1, p27kip1, and p53 were detected by western blot analysis in both YCC-2 and SNU-216 gastric cancer cells. β-actin was used loading control. Cellular senescence was detected by SA-β-galactosidase assay in E. YCC-2 and F. SNU-216 gastric cancer cells. The graph shows the percentage of SA-β-galactosidase-positive cells.
Figure 6
Figure 6. Depletion effect of galectin-3 on the overexpression of hTERT in both the cell proliferation and cellular senescence of human foreskin fibroblasts
A. Human Foreskin fibroblasts were transfected with scRNA alone, pcDNA3.0-hTERT alone, pcDNA3.0-hTERT and galectin-3 siRNA, and galecitn-3 siRNA alone for 48 hr. Each cell plate was stained with Crystal violet solution and showed by photographs (left panel), and cell proliferation in each cell plate was measured by WST assay and presented by quantitative graphs (right panel). B. Detection of mRNA expression levels of hTERT, galectin-3, and p27kip1 were detected by RT-PCR analysis and protein expression levels of these were detected by western blot analysis in human Foreskin fibroblasts. β-actin was used loading control. C. Cellular senescence was detected by SA-β-galactosidase activity in human Foreskin fibroblasts. The graph shows the percentage of SA-β-galactosidase-positive cells.
Figure 7
Figure 7. Detection of interaction between hTERT and galectin-3 through the N-terminal domain of galectin-3 and increased telomeric activity after galectin-3 overexpression
A. Interaction between galectin-3 and hTERT was performed by Immunoprecipitation (IP) assay in HEK293 cells. IP assay was described in “Materials and Methods”. B. Schematic model of galectin-3 delayed mutants; Flag-galectin-3 domains (1–250 aa as the full length; 13-250, 33–250, 63–250, and 111–250 aa. C. Each deleted mutants of galectin-3 and HA-hTERT were co-transfected in HEK293 cells. IP assay with Flag and HA antibodies to detect the interaction of hTERT with galectin-3 domains were performed D. Telomere Repeat Amplification Protocol (TRAP) Assay with galectin-3 binding domain (Full length of galectin-3), galectin-3 wild type, galectin-3 non-binding domain (galectin-3 111–250 AA), and HA-hTERT. TRAP assay was described in “Material and Methods”.
Figure 8
Figure 8. Knockdown of galectin-3 reduces tumor burden in gastric cancer cell xenografted mice, effect that is reversed by overexpression of hTERT
A-D. Lentivirus expressing galectin-3 specific shRNA or retrovirus overexpressing hTERT was prepared and their employed stable YCC-2 gastric cancer cells were established for in vivo study. Lentivirus expressing LacZ shRNA was used as a control and retrovirus overexpressing a vector targeting pBABE-control was used as a control. Mice (n = 5 per group) were inoculated subcutaneously into both flanks with 1 × 106 cells of each YCC-2 cells. A. hTERT overexpression and galectin-3 depletion were confirmed by RT-PCR analysis. β-actin was used loading control. B-C. Photographs B. and quantification of tumor formation C. was performed by measuring tumor size and weight 30 days after inoculation. The error bars indicate 95% confidence intervals: *, P = 0.001; **, P = 0.0015, two-sided t-test. All statistical tests were two-sided. D. Immunohistochemistry (IHC) analysis was performed to detect the expression level of galectin-3 and hTERT in tumors in in vivo mouse models. Method of IHC analysis was described in “Materials and Methods”. Scale bar presents 100 μM.

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