FAM46B inhibits cell proliferation and cell cycle progression in prostate cancer through ubiquitination of β-catenin

Abstract

FAM46B is a member of the family with sequence similarity 46. Little is known about the expression and functional role(s) of FAM46B in prostate cancer (PC). In this study, the expression of FAM46B expression in The Cancer Genome Atlas, GSE55945, and an independent hospital database was measured by bioinformatics and real-time PCR analysis. After PC cells were transfected with siRNA or a recombinant vector in the absence or presence of a β-catenin signaling inhibitor (XAV-939), the expression levels of FAM46B, C-myc, Cyclin D1, and β-catenin were measured by western blot and real-time PCR. Cell cycle progression and cell proliferation were measured by flow cytometry and the CCK-8 assay. The effects of FAM46B on tumor growth and protein expression in nude mice with PC tumor xenografts were also measured. Our results showed that FAM46B was downregulated but that β-catenin was upregulated in patients with PC. FAM46B silencing promoted cell proliferation and cell cycle progression in PC, which were abrogated by XAV-939. Moreover, FAM46B overexpression inhibited PC cell cycle progression and cell proliferation in vitro and tumor growth in vivo. FAM46B silencing promoted β-catenin protein expression through the inhibition of β-catenin ubiquitination. Our data clearly show that FAM46B inhibits cell proliferation and cell cycle progression in PC through ubiquitination of β-catenin.

Fig. 1. FAM46B expression in PC tissues and cell lines

The mRNA expression of FAM46B in PC tissues and corresponding normal prostate tissues collected from an independent hospital a, TCGA b, and the GSE55945 c data set was measured by real-time PCR and bioinformatics analysis. The FAM46B mRNA and protein expression in PC cell lines and prostate epithelial cell lines was measured by real-time PCR d and western blotting e

Fig. 2. Construction of FAM46B RNAi and overexpressing cell lines

Real-time PCR and western blotting were performed to examine the mRNA and protein expression of FAM46B in P69 cells a, b transfected with siFAM46B-1, siFAM46B-2, or siFAM46B-3, and in LNCaP c, d and PC-3 cells e, f transfected with pLVX-Puro-FAM46B. **P < 0.01, ***P < 0.001 compared with siNC or blank vector

Fig. 3. FAM46B silencing promoted cell proliferation and cell cycle progression in P69 cells

After P69 cells were transfected with siFAM46B-1 and siFAM46B-2, cell proliferation, cell cycle progression and the expression of C-myc, Cyclin D1, and β-catenin were measured by CCK-8 assay a, flow cytometry b, c, and western blotting d, respectively. **P < 0.01, ***P < 0.001 compared with siNC

Fig. 4. FAM46B overexpression inhibited cell proliferation and cell cycle progression in LNCaP and PC-3 cells

After LNCaP and PC-3 cells were transfected with pLVX-Puro-FAM46B, cell proliferation, cell cycle progression and the expression of C-myc, Cyclin D1, and β-catenin were measured by CCK-8 assay a, b, flow cytometry c–f, and western blotting g, h, respectively. **P < 0.01, ***P < 0.001 compared with blank vector

Fig. 5. FAM46B overexpression inhibited PC tumor growth in vivo

After LNCaP cells were transfected with pLVX-Puro-FAM46B virus, they were injected into nude mice to establish a xenograft model; after 33 days, the tumor weight a, b and volume c were evaluated, and the protein expression of FAM46B, C-myc, Cyclin D1, and β-catenin were measured in xenograft tumors d, e by western blotting. *P < 0.05, ***P < 0.001 compared with blank vector

Fig. 6. XAV-939 treatment inhibited FAM46B silencing-induced PC cell proliferation and cell cycle process

After P69 cells were transfected with siFAM46B-1 with or without XAV-939 treatment (20 μm), cell proliferation and cell cycle progression were measured by CCK-8 assay a and flow cytometry b, c, and the expression of C-myc, Cyclin D1, and β-catenin was measured by western blotting d, e. PC-3 cells transfected with pLVX-Puro-FAM46B were treated with MG132 (50 μm) for 4 h, and the expression of FAM46B and β-catenin proteins was measured by western blotting f, g. β-catenin was immunoprecipitated and immunoblotted in P69 cells transfected with siFAM46B-1 with or without MG132 (50 μm) treatment for 4 h h. *P < 0.05, **P < 0.01, ***P < 0.001 compared with siNC or blank vector. ^###P < 0.001 compared with siFAM46B-1 or FAM56B

Fig. 7. FAM46B was negatively correlated with β-catenin in PC tissues

The mRNA and protein expression of β-catenin in PC tissues and corresponding normal prostate tissues was measured by real-time PCR a and western blottingb. Linear regression showed that FAM46B mRNA expression was negatively correlated with the mRNA expression of β-cateninc. N1-8: normal prostate tissues; T1-8: PC tissues