UBE2T promotes nasopharyngeal carcinoma cell proliferation, invasion, and metastasis by activating the AKT/GSK3β/β-catenin pathway

Abstract

Increasing evidence has shown that UBE2T plays an important role in genomic integrity and carcinogenesis; however, its role in nasopharyngeal carcinoma (NPC) has not been investigated. Here, we evaluated the clinicopathological significance of UBE2T in NPC and its underlying mechanisms. Using immunohistochemical analysis of UBE2T expression in NPC samples, we demonstrated that UBE2T is highly expressed in NPC tissues, which correlated with the T/M classification, skull invasion, and poor prognosis. The in vitro assay showed that UBE2T overexpression promoted proliferation, migration, and invasion of NPC cells, while UBE2T knockdown inhibited these processes. Consistent with our in vitro results, in vivo studies indicated that UBE2T overexpression promoted the growth of NPC xenografts and NPC cell metastasis. We found that UBE2T overexpression activated, whereas UBE2T knockdown inhibited, the AKT/GSK3β/β-catenin pathway. Moreover, the pathway-activation and in vitro pro-metastasis effects of UBE2T were blocked by the AKT inhibitor, MK-2206 2HCl. Additionally, UBE2T and p-GSK3 β co-expressed in NPC samples by serial section, and their expressions are correlated. Collectively, our findings demonstrated that UBE2T is a possible diagnostic/prognostic biomarker for NPC and may promote the development and progression of NPC by activating the AKT/GSK3β/β-catenin pathway. Thus, UBE2T could serve as an alternative target for the treatment of NPC.

Keywords: AKT/GSK3β/β-catenin pathway; UBE2T; metastasis; nasopharyngeal carcinoma; proliferation.

Figure 1. UBE2T expression in NPC patient samples by IHC

A. Representative image of normal nasopharyngeal mucosal tissue (i) Representative images of nasopharyngeal carcinoma tissue (ii and iii) and a typical cancer nest (iv). Scales indicate 100 μm. B. UBE2T expression in tumor tissue and adjacent normal tissue by IHC (Chi-square test, ***P<0.001). C. Kaplan–Meier survival curve of 149 patients based on the level of UBE2T expression in tumor tissue.

Figure 2. UBE2T promoted NPC cell proliferation in vitro and in vivo

A. Background expression of UBE2T in NPC cell lines was determined by western blotting (left). UBE2T overexpression or knockdown in NPC cells was verified at 48 hours after transfection by western blotting (right). B. and C. MTT assay determined the effects of UBE2T overexpression on C666-1 cell proliferation capacities (B). CCK-8 assay determined the effect of UBE2T knockdown on CNE2 cell proliferation capacities (C). Graph shows the mean ± standard error of mean (SEM) of absorbance at different time points (n=5 or 3, analysis of variance [ANOVA] of factorial design). D. Representative images of colony formation assay (left). The bar chart represents mean ± SEM values for number of colonies from indicated groups (n=3, Student's t-test, ***P<0.001). E. Images of nude mice at indicated time points after subcutaneously injecting C666-1 cells as shown, using the IVIS Lumina II system (up; left flank: UBE2T, right flank: normal control [NC]). The graph shows mean ± SEM values of luminescence signal intensities from both flanks of nude mice at indicated time points (down; n=7, Repeated ANOVA). F. Theimages of indicated C666-1 subcutaneous xenografts (left). The graph shows mean ± SEM values of xenograft weight (n=7, Student's t-test, *P=0.024). G. Representative images of UBE2T and ki-67 expression in C666-1 xenografts from indicated groups by IHC (left). Scales indicate 100 μm. The graph shows mean ± SEM values for the number of ki-67-positive cells from 5 random 40X objective fields for each group (right; Student's t-test, ***P<0.001).

Figure 3. UBE2T promotes NPC cell migration, invasion, and metastasis in vitro and in vivo

A. Representative images of scratch assay from indicated groups at different time points (left); The bar chart represents mean ± SEM of relative scratch width (relative to that at 0 hour width; n=5, ANOVA, *P<0.05, and ***P<0.001). B. Representative images of the matrix-coated transwell assay from indicated groups (left). The bar chart represents mean ± SEM number of invasive cells from 5 random 20X objective fields (Student's t-test, ***P<0.001). C. Images of nude mice photographed with IVIS Lumina II system at 17 days after injecting C666-1 cells in the tail vein. The bar chart represents mean ± SEM of log₂ total luminescence signal intensities (n=7, Student's t-test, *P=0.034).

Figure 4. UBE2T promotes NPC cell proliferation and metastasis probably by activating the AKT/GSK3β/β-catenin pathway

A. Western blot detected the effects of UBE2T on β-catenin, its downstream proliferation/metastasis-related target proteins (Cyclin D1, C-MYC, C-JUN, MMP2, and MMP9), and its upstream pathway proteins (p-AKT, p-GSK3β). B. Immunofluorescence determined the effects of UBE2T overexpression on nuclear translocation of β-catenin. Scales indicate 40 μm. (up; ×1000 field), and separate nuclear and cytoplasmic protein western blot verified the effects of UBE2T on nuclear translocation of β-catenin (down). C. and D. Transwell and matrix-coated transwell analysis detected the effects of AKT inhibitor (MK-2206 2HCl) on the pro-migration and invasion abilities. Representative images of the transwell (C) and matrix-coated transwell (D) assay from indicated groups at 6h (migration) and 24h (invasion). The bar chart represents mean ±SEM number of migration and invasive cells from 5 random 20X objective fields (analysis of variance [ANOVA] of factorial design, ***P<0.001). E. Western blotting detected the effects of AKT inhibitor (MK-2206 2HCl) on AKT/GSK3β/β-catenin pathway. F. UBE2T and P-GSK3β co-expression analysis in the additional NPC samples through serial section technique. Scales indicate 100 μm.