UBE2L3 promotes lung adenocarcinoma invasion and metastasis through the GSK-3β/Snail signaling pathway

Abstract

Lung cancer is the leading cause of cancer-related mortality, and the deaths are mostly attributed to distant metastasis. Previous studies have demonstrated that ubiquitin-conjugating enzyme E2 L3 (UBE2L3) mediates the progression of many human cancers. However, the roles and molecular mechanisms of UBE2L3 in invasion and metastasis of lung adenocarcinoma (LUAD) are yet to be fully understood. Here, we studied the expression pattern of UBE2L3 and demonstrated that it is dramatically up-regulated in LUAD tissues compared with the normal tissues, and its overexpression is positively correlated with lymph node metastasis. Moreover, the upregulation of UBEE2L3 in LUAD tissues is associated with shorter overall survival (OS). UBE2L3 silencing impairs the metastatic capacity of LUAD cells in vitro and in vivo, while its overexpression confers an opposite effect. In addition, our data showed that UBE2L3 promotes cancer cells epithelial-mesenchymal transition (EMT) and metastasis via the glycogen synthase kinase 3β (GSK-3β)/Snail axis. Besides, UBE2L3 was shown to promote ubiquitination and degradation of the GSK-3β. Immunohistochemical analysis demonstrated that UBE2L3 expression is positively correlated with Snail, but negatively correlated with GSK-3β and E-cadherin in LUAD tissues. Taken together, our findings demonstrated that UBE2L3 modulates metastasis of LUAD cells.

Keywords: EMT; GSK-3β; Lung adenocarcinoma; Snail; metastasis.

Figure 1

High UBE2L3 expression correlates with metastasis and poor prognosis in LUAD. The expression profiling and survival analysis in the TCGA-LUAD dataset. A. Box plot analysis of the UBE2L3 mRNA expression in LUAD tissues and normal tissues from the TCGA. B. Kaplan-Meier analysis of the overall survival in LUAD patients based on the expression of UBE2L3. C. Representative immunohistochemistry (IHC) images of UBE2L3 expression in the normal tissues and LUAD tissues with or without lymph node metastasis. Magnification: 40× (scar bar =200 μm) and 200× (scar bar =50 μm). D. H-score was calculated by multiplying the intensity score and the percentage of positive cells (range, 0-300). UBE2L3 was significantly overexpressed in LUAD tissues with lymph node metastasis compared to the LUAD tissues without lymph node metastasis. LN-, lymph node metastasis negative; LN+, lymph node metastasis positive; ***P<0.001.

Figure 2

Depletion of UBE2L3 inhibits metastasis of LUAD cells. A. Immunoblotting analyses were performed to determine the expression of UBE2L3 in LUAD cells. B. Representative images of wound healing assays and the percentage of wound closure. Magnification: 40× (scar bar =200 μm). C. The effect of UBE2L3 konckdown on the migration and invasion of LUAD cells was assessed using Transwell assays. Magnification: 100× (scar bar =100 μm). D, E. Representative HE staining of lung metastasis after the injection of cancer cells into BALB/c mice via tail vein. Magnification: 100× (scar bar =100 μm). Quantification of lung metastasis (n=5 mice per group). ***P<0.001; **P<0.001.

Figure 3

Overexpression of UBE2L3 promotes tumor cell metastasis. A. Immunoblotting analyses were performed to determine the expression of UBE2L3 in LUAD cells. B. Representative images of wound healing assays and the percentage of wound closure. Magnification: 40× (scar bar =200 μm). C. The effect of UBE2L3 overexpression on the migration and invasion of LUAD cells was assessed using Transwell assays. Magnification: 100× (scar bar =100 μm). D, E. Representative HE staining of lung metastasis after the injection of cancer cells into BALB/c mice via tail vein. Magnification: 100× (scar bar =100 μm). The quantification of lung metastasis (n=5 mice per group). ***P<0.001; **P<0.001.

Figure 4

UBE2L3 interacts with and ubiquitinates GSK-3β. (A) GSK-3β levels were determined through immunoblotting and qRT-PCR. (B) H1299/UBE2L3 and the control cells were treated with 100 μg/ml CHX. The cells were harvested and subjected to immunoblotting at different times. Quantification of the UBE2L3 levels, which were normalized to GAPDH levels at 0 h. (C, D) 293T cells were co-transfected with Flag-GSK-3β and myc-UBE2L3 plasmids. Reciprocal co-immunoprecipitation of myc-UBE2L3 and Flag-GSK-3β in co-transfected 293T cells. Endogenous UBE2L3 interacted with GSK-3β in H1299 cells (E) and A549 cells (F). Cells were treated with 20 μM MG132 for 4 h. The cells were harvested and subjected to immunoblotting analyses with anti-Ubiquitin antibodies. UBE2L3 overexpression dramatically promoted the ubiquitination of GSK-3β in H1299 cells (G) and A549 cells (H).

Figure 5

UBE2L3 up-regulates Snail expression in a GSK-3β dependent manner. A. The effect of UBE2L3 expression on the EMT markers (Snail and E-cadherin) was determined by immunoblotting. B. Immunoblotting analyses were performed to assess the effect of the GSK-3β inhibitor (CHIR 99021) on EMT makers.

Figure 6

UBE2L3 is dependent on elevated Snail to enhance migration and invasion of LUAD cells. A. The efficiency of Snail knockdown in H1299 cells was assessed by immunoblotting. B. Representative images of wound healing assays and the percentage of wound closure were quantified. H1299 cells were co-transfected with the UBE2L3 expression vector and Snail shRNA or control shRNA. Snail knockdown suppressed the cell migration induced by UBE2L3. Magnification: 40× (scar bar =200 μm). C. Cells were analyzed by Transwell assays. Magnification: 100× (scar bar =100 μm). D. Knockdown of Snail inhibited the lung metastasis induced by UBE2L3 in vivo. Representative HE staining of lung metastasis after injection of cancer cells into BALB/c mice via the tail vein. Magnification: 100× (scar bar =100 μm). Quantification of lung metastasis by section of the whole lung (n=5 mice per group). ***P<0.001.

Figure 7

Clinical significance of UBE2L3/GSK-3β/Snail crosstalk in LUAD and schematic diagram. A. Representative immunohistochemical images of LUAD specimens stained with UBE2L3, GSK-3β, Snail, or E-cadherin antibodies. Magnification: 200× (scar bar =50 μm). B. Quantification of the immunohistochemistry analyses. Chi-square test was used to analyze the relationship between UBE2L3 and Snail, GSK-3β, or E-cadherin. **P<0.01; *P<0.05. C. The schematic diagram shows that UBE2L3 promotes LUAD cell migration and invasion by inducing EMT through the GSK-3β/Snail axis.