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. 2017 Aug 24;8(48):84193-84203.
doi: 10.18632/oncotarget.20449. eCollection 2017 Oct 13.

Ubiquitin conjugating enzyme E2 L3 promoted tumor growth of NSCLC through accelerating p27kip1 ubiquitination and degradation

Xingjie Ma  1 , Junjie Zhao  1 , Fan Yang  1 , Haitao Liu  1 , Weibo Qi  1
Affiliations

Ubiquitin conjugating enzyme E2 L3 promoted tumor growth of NSCLC through accelerating p27kip1 ubiquitination and degradation

Xingjie Ma et al. Oncotarget. .

Abstract

The molecular pathogenesis of human lung cancer has not been completely clarified. Here, we reported that UBE2L3, a member of the ubiquitin-conjugating enzymes (E2s), were overexpressed in non-small-cell lung cancer (NSCLC) tissues compared with the non-tumor tissues. High expression of UBE2L3 was correlated with advanced tumor stage and adverse outcomes. Knockdown of UBE2L3 inhibited NSCLC cell growth while ectopic expression of UBE2L3 promoted NSCLC cell growth in a cell cycle dependent manner. The results of subcutaneous tumor xenograft studies revealed that knockdown of UBE2L3 attenuated the in vivo tumor growth. Mechanistically, we observed that UBE2L3 could interact with F-box protein Skp2, a member of the SCF (Skp2) ubiquitin ligase complex, and thus promoted the ubiquitination and proteasomal degradation of p27kip1. Furthermore, NSCLC cases with high level of UBE2L3 and low level of p27kip1 had worst prognosis, suggesting that combination of UBE2L3 and p27kip1 is a more powerful prognostic marker for NSCLC patients. Taken together, the current study presented a novel marker for predicting prognosis and a potential therapeutic target for NSCLC patients.

Keywords: NSCLC; UBE2L3; degradation; p27kip1; ubiquitination.

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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 UBE2L3 and its clinical significance in NSCLC patients
(A) Representativeimmunoblotting (IB) images of UBE2L3 expression in 28 paired NSCLC samples. (B) Quantification of relative grey value of UBE2L3 compared with GAPDH. (C) The protein level of UBE2L3 was detected by IB in eight NSCLC cell lines and the normal immortalized bronchial epithelial cell line HBE. (D) Representative images of the immunohistochemical staining for UBE2L3 in tumor tissues and non-tumor tissues. (E) NSCLC patients with high level of UBE2L3 showed worse overall survival compared with the cases exhibited low level of UBE2L3. (Data were presented with mean ± SD of three independent experiments, **p<0.01).
Figure 2
Figure 2. Knockdown of UBE2L3 inhibited cell cycle dependent proliferation of NSCLC cells
(A, C) Knockdown efficiency of UBE2L3 in A549 and NCI-H1299 cells were confirmed by IB. (B, D) Knockdown of UBE2L3 inhibited cell proliferation of A549 and NCI-H1299 cells by CCK-8 assay. (E, F) Knockdown of UBE2L3 inhibited colony formation ability of A549 and NCI-H1299 cells. (G, H) Cell cycle of NSCLC cells was detected by flow cytometry and the results indicated that knockdown of UBE2L3 increased proportion of cells in G1 phase. (Data were presented with mean ± SD of three independent experiments, *p<0.05, **p<0.01).
Figure 3
Figure 3. UBE2L3 interacted with SCF (Skp2) complex and promoted p27kip1 ubiquitination and proteasomal degradation
(A) Knockdown of UBE2L3 in A549 and NCI-H1299 cells promoted the protein level of p27kip1. (B) Overexpression of UBE2L3 in NCI-H460 and HCC827 cells decreased the protein level of p27kip1. (C) The association between UBE2L3 and SCF (Skp2) complex and p27kip1 was confirmed by immunoprecipitation and immunoblotting assay. (D) Overexpression of UBE2L3 promoted the ubiquitination and proteasomal degradation of p27kip1. (E, F) The half-life of p27kip1 was evaluated by CHX assay after UBE2L3 overexpression.
Figure 4
Figure 4. p27kip1 mediated UBE2L3's function in NSCLC cells
(A, B) Knockdown of UBE2L3 in A549 and NCI-H1299 cells promoted the level of p27kip1 and inhibited NSCLC cell growth, while co-transfection of siUBE2L3 and sip27kip1 downregulated the level of p27kip1 and promoted NSCLC cell growth. (C, D) Overexpression of UBE2L3 decreased the level of p27kip1 and promoted NSCLC cell growth, while co-transfection of UBE2L3 and p27kip1 expressing plasmids promoted the level of p27kip1 and inhibited NSCLC cell growth. (Data were presented with mean ± SD of three independent experiments, **p<0.01).
Figure 5
Figure 5. UBEL23 and p27kip1 could be used as prognostic biomarkers together
(A, B) Representative immunohistochemicalimages of UBEL23 and p27kip1. (C) Correlation analysis of UBEL23 and p27kip1 expression were presented. (D) High level of p27kip1 predicted better overall survival (OS) of NSCLC patients. (E) NSCLC cases expressing high level of UBE2L3 and low level of p27kip1 had the poorest prognosis in terms of OS.
Figure 6
Figure 6. UBE2L3 contributes to NSCLC progression in vivo
(A, D) A549 and NCI-H1299 cells stably expressing shUBE2L3 or control vector were subcutaneously transplanted into nude mice and the growth curves were shown. (B, E) The weights of tumors were presented as mean ± SD. (C, F) IHC staining demonstrated that knockdown of UBE2L3 inhibited NSCLC cell growth in vivo, as indicated by the expression of ki67. (Data were presented with mean ± SD of three independent experiments, **p<0.01).
Figure 7
Figure 7. Schematic diagram summarizing how UBE2L3 regulated cell cycle through accelerating SCF (Skp2) mediated p27kip1 degradation
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