Cyclin G2 suppresses Wnt/β-catenin signaling and inhibits gastric cancer cell growth and migration through Dapper1

Abstract

Background: Gastric cancer is one of the most common malignant tumors. Cyclin G2 has been shown to be associated with the development of multiple types of tumors, but its underlying mechanisms in gastric tumors is not well-understood. The aim of this study is to investigate the role and the underlying mechanisms of cyclin G2 on Wnt/β-catenin signaling in gastric cancer.

Methods: Real-time PCR, immunohistochemistry and in silico assay were used to determine the expression of cyclin G2 in gastric cancer. TCGA datasets were used to evaluate the association between cyclin G2 expression and the prognostic landscape of gastric cancers. The effects of ectopic and endogenous cyclin G2 on the proliferation and migration of gastric cancer cells were assessed using the MTS assay, colony formation assay, cell cycle assay, wound healing assay and transwell assay. Moreover, a xenograft model and a metastasis model of nude mice was used to determine the influence of cyclin G2 on gastric tumor growth and migration in vivo. The effects of cyclin G2 expression on Wnt/β-catenin signaling were explored using a TOPFlash luciferase reporter assay, and the molecular mechanisms involved were investigated using immunoblots assay, yeast two-hybrid screening, immunoprecipitation and Duolink in situ PLA. Ccng2^-/- mice were generated to further confirm the inhibitory effect of cyclin G2 on Wnt/β-catenin signaling in vivo. Furthermore, GSK-3β inhibitors were utilized to explore the role of Wnt/β-catenin signaling in the suppression effect of cyclin G2 on gastric cancer cell proliferation and migration.

Results: We found that cyclin G2 levels were decreased in gastric cancer tissues and were associated with tumor size, migration and poor differentiation status. Moreover, overexpression of cyclin G2 attenuated tumor growth and metastasis both in vitro and in vivo. Dpr1 was identified as a cyclin G2-interacting protein which was required for the cyclin G2-mediated inhibition of β-catenin expression. Mechanically, cyclin G2 impacted the activity of CKI to phosphorylate Dpr1, which has been proved to be a protein that acts as a suppressor of Wnt/β-catenin signaling when unphosphorylated. Furthermore, GSK-3β inhibitors abolished the cyclin G2-induced suppression of cell proliferation and migration.

Conclusions: This study demonstrates that cyclin G2 suppresses Wnt/β-catenin signaling and inhibits gastric cancer cell growth and migration through Dapper1.

Keywords: Cyclin G2; Dpr1; Gastric Cancer; Tumor suppressor; Wnt/β-catenin signaling.

Fig. 1

Cyclin G2 expression is down-regulated in gastric cancer. a Relative mRNA expression of CCNG2 in 45 paired gastric cancer and adjacent normal tissues. The quantification of CCNG2 mRNA was performed using qRT-PCR. Data are presented as log₂ values in gastric cancer tissues relative to matched adjacent non-tumor tissues. b Western blot analysis for the cyclin G2 protein levels in ten gastric cancer tissues and adjacent non-cancerous gastric tissues. c Representative immunohistochemical staining with cyclin G2 antibody in gastric cancer and paired adjacent normal tissues. Six representative cases are shown. Scale bar = 100 μm. d Relative mRNA expression of CCNG2 in GES-1 and three gastric cancer cell lines (SGC-7901, AGS and MGC-803) with different differentiation statuses. CCNG2 mRNA was quantified by qRT-PCR. Data from gastric cancer cell lines are expressed relative to the GES-1 CCNG2 mRNA level (a normal gastric epithelial cell line that was chosen as a control). Results are presented as the means of values. Bars indicate standard deviation (SD; n = 3). *P < 0.05; **P < 0.01. e Comparison of the protein expression levels of cyclin G2 in three gastric cell lines and human normal gastric epithelial cell line

Fig. 2

Cyclin G2 inhibits the proliferation and migration of SGC-7901 or MGC-803 gastric cancer cells. a Western blot analysis for the cyclin G2 protein levels in cyclin G2 overexpression and knockdown SGC-7901 or MGC-803 cells. b MTS cell proliferation assay in cyclin G2 overexpression and cyclin G2 knockdown SGC-7901 or MGC-803 cells (n = 3).*P < 0.05; **P < 0.01. c and d Colony formation assay in cyclin G2 overexpression and cyclin G2 knockdown SGC-7901 or MGC-803 cells (n = 3).*P < 0.05; **P < 0.01. e and f Representative images and quantification of cell cycle analysis after cyclin G2 overexpression in SGC-7901 cells by flow cytometry. g and h Representative images and quantification of the wound healing assay in cyclin G2 overexpression and cyclin G2 knockdown SGC-7901 or MGC-803 cells (n = 3).*P < 0.05; **P < 0.01. i and j Representative Transwell® migration assays in cyclin G2 overexpression SGC-7901 or MGC-803 cells determined by stained with Giemsa. The migration ratio is expressed as a percentage of the control cells at the indicated time-points (n = 3). **P < 0.01

Fig. 3

Cyclin G2 inhibited tumor growth and metastasis in vivo. a Western blot analysis for the cyclin G2 expression in SGC-7901 cells (blank), SGC-7901 cells overexpressing cyclin G2 and GFP (NC). b–d Cyclin G2 inhibited tumor growth in the xenograft nude mice. Nude mice were subcutaneously injected with SGC-7901 cells (blank), SGC-7901 cells overexpressing cyclin G2 or control GFP (NC) by lentivirus infection. Tumor sizes were measured after sizeable tumors formation (day 14). Inoculated mice were sacrificed on day 25 and the tumors were excised for analysis (n = 5). b Macroscopic appearance of the isolated tumor. c The tumor weights in cyclin G2-overexpressing, control vector GFP-overexpressing (NC) and untransfected SGC-7901 cell (blank) groups were compared (n = 5). *P < 0.05. d Comparison of tumor volume curves in uninfected SGC-7901 cells (blank), SGC-7901 cells overexpressing cyclin G2 and GFP (NC) groups in xenograft nude mice (n = 5). e Representative immunohistochemical staining with a Ki-67 antibody of the xenograft tumor from nude mice injected with SGC-7901 negative control cells and SGC-7901 cells overexpressing cyclin G2. Bar = 100 μm. f Graph represents percentage of Ki-67-positive cells out of the total cells in each field (n = 3). **P < 0.01. g Representative immunohistochemical staining of xenograft tumors from nude mice with cyclin G2 antibody. Bar = 100 μm. h–i The effect of cyclin G2 on gastric cancer metastasis in vivo. (h) Representative images of whole lungs from nude mice inoculated with SGC-7901 cells overexpressing cyclin G2 or control GFP (NC) by lentivirus infection. (i) Statistical analysis showed a decrease in the metastatic foci of the nude mice inoculated with SGC-7901 cells overexpressing cyclin G2 compared to those overexpressing GFP. *P < 0.05. j Representative H&E stain images of Lung metastasis. Bar = 100 μm

Fig. 4

Cyclin G2 inhibits Wnt/β-catenin signaling by promoting β-catenin degradation in a GSK-3β dependent manner. a β-catenin transcriptional activity of SGC-7901 and MGC-803 cells overexpressing cyclin G2 or control vector (NC) was determined by a TOPFlash luciferase reporter assay (n = 3). *P < 0.05. b Western blot analysis for the β-catenin expression in ectopic cyclin G2 overexpression in SGC-7901 or MGC-803 cells compared with the negative vector control. c TOPFlash luciferase reporter assays showed that ectopic cyclin G2 suppressed β-catenin transcriptional activity in HeLa, HT-29, HEK-293, and COS-7 cells. d COS-7 cells infected with incremental concentrations of recombinant retroviruses encoding CCNG2 (Re-cyclin G2) or a negative control gene were used for western blotting after 48 h incubation. Western blot analysis showed the overexpression of cyclin G2 inhibited β-catenin and c-Myc expression in a dose-dependent manner. e β-catenin and c-Myc expression levels in Ccng2^−/− and WT MEFs were analyzed by western blot. f Western blot analysis revealed that the expression and phosphorylation of β-catenin and GSK-3β in the negative control and cyclin G2-overexpressing COS-7 cells. g Western blot analysis for β-catenin protein levels in the control or cyclin G2-overexpressing COS-7 cells treated with or without GSK-3β inhibitor, LiCl (20 mM), for 24 h. NaCl was used as a negative control. h Western blot analysis to determine the β-catenin protein levels in the control or cyclin G2-overexpressing COS-7 cells treated with or without proteasome inhibitor, MG132 (25 μM), for 6 h. DMSO was used as a negative control

Fig. 5

Cyclin G2 inhibits Wnt/β-catenin signaling through DPR1. a Cell lysates from COS-7 cells were immunoprecipitated with Dpr1 or IgG control antibody, followed by western blot analysis with a cyclin G2 antibody (upper panel). Total cellular protein from COS-7 cells was immunoprecipitated with an anti-cyclin G2 or IgG control antibody, then blotted with anti-DPR1 and anti-Dvl2 antibody, respectively (middle panel). Western blot analysis of endogenous Dpr1 and cyclin G2 protein level in COS-7 cells (lower panel). b Direct interaction of Dpr1 and cyclin G2 was detected using Duolink in situ PLA. Red spots represent the interaction of Dpr1 and cyclin G2. The nuclei were stained using DAPI and are shown in blue. Images were acquired using a confocal microscopy with a 40× objective. c Western blot analysis of the Dvl2 protein levels in the control or cyclin G2-overexpressing COS-7 cells treated with 25 μM MG132, 1 μM bafilomycin A1 (BFA1), or DMSO as a negative vehicle control for 6 h followed. d Overexpression of cyclin G2 down-regulated β-catenin and Dvl2 protein expression levels in COS-7 cells, whereas knockdown of Dpr1 attenuated the effect of cyclin G2. cyclin G2 overexpressing vector or control vector (NC) was co-transfected with Dpr1-specific (shDACT1) or nonspecific shRNA vectors (shNC) into COS-7 cells for 48 h followed by western blot analysis. e The phosphorylation level of Dpr1 was suppressed by cyclin G2. Cell lysates from HEK-293 and SGC-7901 cells overexpressing cyclin G2 or GFP as a negative control was immunoprecipitated with anti-Dpr1 antibodies, then immunoblotted with anti-Phosphoserine/threonine antibody (upper panel). Western blot analysis of Dpr1 and cyclin G2 expression in total cellular protein (lower panel). f Cyclin G2 inhibited the CKI-induced phosphorylation of Dpr1. HEK-293 cells were transfected with expression vectors encoding CKI (pEGFP-CKI) and cyclin G2 (pCMV-3 × FLAG-G2) or transfected with negative control vectors (pCMV-3 × FLAG-BAP or pEGFP-N3) for 48 h. The cell lysates were immunoprecipitated with Dpr1 antibodies and immunoblotted with anti-Phosphoserine antibody (upper panel). Dpr1 and cyclin G2 expression in total cellular protein was analysed by western blot (lower panel)

Fig. 6

Cyclin G2 inhibits the gastric cancer proliferation and migration hrough Wnt/β-catenin signaling. a-e SGC-7901 cells were infected with cyclin G2 recombinant lentiviruses (LV-cyclin G2) or negative control lentivirus (LV-GPF) for 42 h and then treated with CHIR99021 or DMSO as a negative vehicle control. a and b The infection efficiency of recombinant lentivirus was confirmed by measurement of GFP vector expression (Original magnification, × 200) and western blot analysis of cyclin G2. c Cell lysates were subjected to immunoblot analysis to measure β-catenin protein expression level. d The cell proliferation of SGC-7901 cells was measured by the MTS assay (n = 6). *P < 0.05; **P < 0.01. e The cell migration of SGC-7901 cells was measured by Transwell^® migration assays. The data were presented as migrated cells as a percentage of the controls cells at the indicated time-points (n = 3). *P < 0.05; **P < 0.01. f-k AGS cells were overexpressed or knockdown of cyclin G2. f Cell lysates were subjected to immunoblot analysis to determine β-catenin protein expression level in cyclin G2 overexpression or knockdown AGS cells compared with negative control. g MTS cell proliferation assay, (i and j) colony formation assay and (k and l) Transwell® migration assay in cyclin G2 overexpression or knockdown AGS cells compared with the negative control (n = 3). h Cell cycle distributions were detected by flow cytometry analysis in cyclin G2 overexpression AGS cells compared with the negative control (n = 3). *P < 0.05; **P < 0.01

Fig. 7

Hypothesied pathway showing the inhibitory mechanism of cyclin G2 on Wnt/β-catenin signaling through Dpr1. a Wnt ligands bind to Fzd and LRP co-receptor complexes, which in turn activates Dvl2. Dvl2 inhibits the activity of GSK-3β triggering β-catenin phosphorylation and preventing its degradation. Stabilized β-catenin activates gene expression of the Wnt/β-catenin signaling pathway and upregulates tumorigenesis. The inhibitory effect of Dpr1 on the Wnt/β-catenin signaling is blocked when phosphorylated by CKI. b Cyclin G2 interacts with Dpr1 and impacts its phosphorylation level, enhancing proteasome-dependent degradation of Dvl2. As a result, increasing the ability of GSK-3β to phosphorylate β-catenin and accelerate its degradation. This leads to suppression of Wnt/β-catenin signaling and inhibition of tumorigenesis of gastric cancer