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. 2017 Aug 24;8(44):77241-77253.
doi: 10.18632/oncotarget.20455. eCollection 2017 Sep 29.

CUL4B promotes bladder cancer metastasis and induces epithelial-to-mesenchymal transition by activating the Wnt/β-catenin signaling pathway

Xia-Wa Mao  1 , Jia-Quan Xiao  1 , Gang Xu  1 , Zhong-Yi Li  1 , Hui-Feng Wu  1 , Yi Li  1 , Yi-Chun Zheng  1 , Nan Zhang  1
Affiliations

CUL4B promotes bladder cancer metastasis and induces epithelial-to-mesenchymal transition by activating the Wnt/β-catenin signaling pathway

Xia-Wa Mao et al. Oncotarget. .

Abstract

Increased expression of cullin 4B (CUL4B) is linked to progression in several cancers. This study aims to explore the effects of CUL4B on bladder cancer (BC) metastasis and epithelial-to-mesenchymal transition (EMT) and potential correlation to the Wnt/β-catenin signaling pathway. We collected BC tissues and adjacent normal tissues from 124 BC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were employed in order to detect the expression of Wnt/β-catenin signaling pathway-related proteins and EMT markers. MTT and Transwell assays were used in order to measure cell proliferation, migration, and invasion. BC 5637 cells were transfected with control, siRNA scramble control (siRNA-NC), si-CUL4B, and CUL4B or Wnt inhibitory factor 1 (WIF-1) overexpression constructs. Levels of CUL4B mRNA and protein were increased in BC tissues in comparison with the adjacent normal tissues. CUL4B expression was negatively correlated with the expression of E-cadherin and positively correlated to the expression of N-cadherin and Vimentin. Compared to the control group, levels of β-catenin, cyclinD1, c-myc, MMP7, and EMT markers were reduced, whereas phosphorylated GSK3βSer9 and E-cadherin levels were increased in the si-CUL4B and WIF-1 groups. In addition, cell proliferation, migration, and invasion abilities were also increased. Increasing CUL4B expression had the opposite effect. These findings suggest that CUL4B induces EMT and promotes metastasis of BC by activating the Wnt/β-catenin signaling pathway.

Keywords: CUL4B; Wnt/β-catenin signaling pathway; bladder cancer; epithelial-to-mesenchymal transition; proliferation.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Expression of CUL4B and EMT marker proteins in BC tissues and adjacent normal tissues
(A) Expression of CUL4B and EMT markers detected by IHC. The percentage of cells expressing CUL4B, E-cadherin, N-cadherin, and Vimentin in the adjacent normal tissues was 2%, 87%, 5%, and 3%, respectively; the staining intensity was 0, 3, 0, and 0, respectively. The percentage of cells expressing CUL4B, E-cadherin, N-cadherin and Vimentin in the BC tissues was 28%, 16%, 70%, and 3%, respectively. The staining intensity was 3, 1, 3, and 3, respectively; (B) the percentage of high expression of CUL4B and EMT marker proteins.
Figure 2
Figure 2. The mRNA and protein expression in BC cell lines EJ, 5637, T24, and RT-4
(A) CUL4B, E-cadherin, N-cadherin, and Vimentin mRNA expression; (B) CUL4B, E-cadherin, N-cadherin and Vimentin protein expression; (C) cartogram of CUL4B, E-cadherin, N-cadherin, and Vimentin protein expression. * compared with cell line 5637, P < 0.05.
Figure 3
Figure 3. Relative mRNA and protein expression of CUL4B in each group
(A) Relative mRNA expression of CUL4B in each group detected by qRT-PCR; (B) protein expression of CUL4B detected by western blotting; (C) relative CUL4B protein expression in each group. * compared with the control group, P < 0.05.
Figure 4
Figure 4. Effect of CUL4B on the Wnt/β-catenin signaling pathway
(A) mRNA expression of β-catenin, cyclinD1, c-myc, and MMP7 in each group determined by qRT-PCR; (B) expression of β-catenin/β-actin, cyclinD1/β-actin, c-myc/β-actin, and MMP7/β-actin in each group determined by western blotting; (C) cartogram of protein expression of β-catenin, cyclinD1, c-myc and MMP7 in each group; (D) mRNA expression of GSK3β in each group determined by qRT-PCR; (E) expression of t-GSK3β/GAPDH, p-GSK3βSer9/GAPDH in each group determined by western blotting; (F) cartogram of levels of p-GSK3βSer9/t-GSK3β in each group. * compared with the control group, P < 0.05.
Figure 5
Figure 5. Effect of CUL4B on EMT in vitro
(A) mRNA expression of E-cadherin, N-cadherin, Vimentin, Snail1, ZEB1, and ZEB2 in each group detected by qRT-PCR; (B) protein expression of E-cadherin, N-cadherin, Vimentin, Snail1, ZEB1, ZEB2, and GAPDH in each group detected by western blotting; (C) cartogram of protein expression of E-cadherin, N-cadherin, Vimentin, Snail1, ZEB1, and ZEB2 in each group. * compared with the control group, P < 0.05.
Figure 6
Figure 6. Effect of CUL4B on BC cell proliferation
* compared with the control group, P < 0.05.
Figure 7
Figure 7. Effect of CUL4B on BC cell migration and invasion
(A) CUL4B interference inhibited the cell migration ability in each group detected by Transwell assay; (B) the number of migrating cells in each group; (C) CUL4B interference inhibited the cell invasion ability in each group detected by Transwell assay; (D) the number of invasive cells in each group. * compared with the control group, P < 0.05.
Figure 8
Figure 8. Effect of CUL4B on tumor growth in nude mice in vivo
(A) Tumor volume in each group; (B) tumor weight in each group. * compared with the control group, P < 0.05.
Figure 9
Figure 9. Effect of CUL4B on EMT in vivo
(A) mRNA expression of E-cadherin, N-cadherin, and Vimentin in nude mouse xenografts determined by qRT-PCR; (B) the protein expression of E-cadherin, N-cadherin, and Vimentin in nude mouse xenografts detected by IHC. * compared with the control group, P < 0.05.
Figure 10
Figure 10. Effect of CUL4B on the number of lung metastases in BC cells in each group
* compared with the control group, P < 0.05.
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