TNFRSF11B activates Wnt/β-catenin signaling and promotes gastric cancer progression

Abstract

Tumor necrosis factor receptor superfamily member 11B (TNFRSF11B) has been studied to be involved in the development and progression of several human malignancies. However, little is unveiled regarding the complex mechanisms of TNFRSF11B in human gastric cancer (GC). The clinical significance of TNFRSF11B was assessed in 70 and 160 GC tissues using immunohistochemistry method and gene microarray analysis, respectively. The biological function of TNFRSF11B was studied in vitro and in vivo assays. Immunofluorescence assay was used to evaluate the expression of β-catenin in the nucleus. The expression of β-catenin and related protein was determined by Western blot. The interaction between TNFRSF11B and GSK3β was detected by co-immunoprecipitation. We demonstrated that TNFRSF11B was highly expressed in the cytoplasm of GC and associated with the patient poor outcome. Our studies showed that TNFRSF11B in GC cells significantly promoted cell proliferation, migration, invasion in vitro and tumorigenic ability in vitro and in vivo. Meanwhile, TNFRSF11B inhibited GC cell apoptosis. The proportion of nuclear active β-catenin showed positively correlation with TNFRSF11B expression. TNFRSF11B directly combined with GSK-3β upregulating its phosphorylation, and increased expression of β-catenin and its downstream effectors. Collectively, these findings demonstrate that TNFRSF11B promote the aggressive phenotypes of GC cells and activated Wnt/β-catenin signaling. Accordingly, TNFRSF11B had potential as a biomarker and inhibition of TNFRSF11B expression might offer a new therapeutic target for GC patients.

Keywords: TNFRSF11B; TRAIL; Wnt/β-catenin; apoptosis; gastric cancer; tumorigenesis.

Figure 1

The expression of TNFRSF11B in gastric cancer. (A) The expression of TNFRSF11B was analyzed in the Cancer Genome Atlas (TCGA) project gastric cancer database, where a significant elevation in TNFRSF11B expression was observed in gastric adenocarcinoma tissues(n=415) compared to normal tissues(n=34). (B) The expression of TNFRSF11B was also analyzed in the Oncomine database where diffuse gastric adenocarcinoma (n=45), gastric adenocarcinoma(n=173), gastric intestinal type adenocarcinoma (n=41), gastric tubular adenocarcinoma (n=31) and mucinous gastric adenocarcinoma (n=12) were all found to be enhanced in comparison to normal tissues(n=330). (C) Expression of TNFRSF11B by immunohistochemical staining. Original magnification: 200×. N: normal stomach; T: gastric cancer (D) Association between TNFRSF11B expression status and TNM stage. (E) Kaplan-Meier survival curves of overall survival for 70 gastric cancer patients with TNFRSF11B-negative vs TNFRSF11B-positive GC tissue by IHC. (F) The expression of TNFRSF11B in primary GC (T) and corresponding surgical margin (N) tissues was examined by gene microarray analysis. (G) Kaplan-Meier survival curves of overall survival (OS) for 160 patients with TNFRSF11B-negative vs. TNFRSF11B-positive by gene microarray analysis.

Figure 2

TNFRSF11B promotes GC cell growth in vitro and in vivo. (A and B) Proliferation activities were higher in HGC-27 and BGC-823 overexpression cells. (C and D) Proliferation activities were lower in MGC-803 and SGC-7901 knockdown cells. (E and F) Up-regulation of TNFRSF11B significantly increased cell clonogenicity in HGC-27 and BGC-823 cells. (G and H) Down-regulation of TNFRSF11B significantly reduced cell clonogenicity in MGC-803 and SGC-7901 cells. (I) Photograph showing tumor formation in nude mice injected with MGC803-Scramble and shTNFRSF11B, as well as tumor weights and tumor growth curve. The data are shown as mean ± SD. *p<0.05, **p<0.01, ***p<0.001

Figure 3

TNFRSF11B promotes GC cell migration and invasion. (A and B) In HGC-27 and BGC-823 overexpression cells, migration activities were significantly higher. (C and D) In MGC-803 and SGC-7901 cells, migration activities were significantly lower in TNFRSF11B knockdown cells. (E and F) TNFRSF11B overexpression increased migration and invasion of HGC-27 and BGC-823 cells in comparison to their respective control cells. (G and H) TNFRSF11B knockdown reduced migration and invasion ability in MGC-803 and SGC-7901 cells.*p<0.05, **p<0.01, ***p<0.001

Figure 4

Effects of TNFRSF11B on apoptosis in GC cells. (A and B) TNFRSF11B overexpression cell groups showed decreased apoptosis rate in HGC-27 and BGC-823 cells. (C and D)TNFRSF11B knockdown cell groups showed increased apoptosis rate in MGC-803 and SGC7901 cells. (E) In TUNEL assay, the apoptotic cell rate in the HGC-27 TNFRSF11B overexpression group was significantly lower than the control group. (F) The apoptotic cell rate in the MGC-803 TNFRSF11B knockdown group was significantly higher than the control group. (G) The expression of BCL-2 was higher and the BAX, PARP and Caspase3 were lower in HGC-27 and BGC-823 TNFRSF11B overexpression cells than control cells. (H) The expression of BCL-2 was low and the BAX、PARP and Caspase3 were high in MGC-803 and SGC-7901 cells. 1: Mock, 2: TNFRSF11B, 3: Scramble, 4: shTNFRSF11B. *p<0.05, **p<0.01, ***p<0.001

Figure 5

TNFRSF11B affected β-catenin activity and the nuclear localization of β-catenin. (A) Overexpression of TNFRSF11B significantly enhanced the transcriptional activity of β-catenin in BGC-823 cells by TOP-Flash reporter assay. (B) Knockdown of TNFRSF11B significantly suppressed the transcriptional activity of β-catenin in MGC-803 cells by TOP-Flash reporter assay. (C and D) The nuclear localization of active β-catenin in four GC cells was detected using immunofluorescent microscopy. The representative images are shown. The proportion of nuclear active β-catenin showed positively correlation with TNFRSF11B expression. *p<0.05, **p<0.01, ***p<0.001

Figure 6

TNFRSF11B increased β-catenin and related protein expression and hypothetic model for the mechanism of TNFRSF11B in gastric cancer. (A) Overexpression of TNFRSF11B increased the protein level of cytoplasmic and nuclear β-catenin in BGC-823 cells. (B) Knockdown of TNFRSF11B decreased the protein level of cytoplasmic and nuclear β-catenin in MGC-803 cells. (C) Overexpression of TNFRSF11B increased β-catenin expression in HGC-27 and BGC-823 cells. (D) Knockdown of TNFRSF11B decreased β-catenin expression in MGC-803 and SGC-7901 cells. (E) Overexpression of TNFRSF11B increased the protein expression of CyclinD1, c-Myc, MMP-7, GSK3β, p-GSK3β in BGC-823 cells. (F) Knockdown of TNFRSF11B decreased the protein expression of CyclinD1, c-Myc, MMP-7, GSK3β, p-GSK3β in SGC-7901 cells. (G) The Co-immunoprecipitation analysis of MGC-803 and SGC-7901 GC cell lines showed mutual interaction between TNFRSF11B and GSK-3β. (H) Hypothetic model for the function and mechanism of TNFRSF11B in gastric cancer. 1:Mock, 2:TNFRSF11B, 3:Scramble, 4: shTNFRSF11B. *p<0.05, **p<0.01, ***p<0.001