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UBE2S enhances the ubiquitination of p53 and exerts oncogenic activities in hepatocellular carcinoma
Biochemical and Biophysical Research Communications xxx (2018) 1e8
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UBE2S enhances the ubiquitination of p53 and exerts oncogenic activities in hepatocellular carcinoma Ying-Hua Pan a, 1, Mei Yang b, 1, Li-ping Liu c, Dan-Chun Wu a, Ming-yue Li c, Shu-Guang Su d, * a
Department of Rheumatology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China Department of Gastroenterology, Dongguan Third People's Hospital, Dongguan, China Department of Hepatobiliary and Pancreatic Surgery, Shenzhen People's Hospital, Second Clinical Medical College of Jinan University, Shenzhen, Guangdong Province, China d Department of Pathology, The Affiliated Hexian Memorial Hospital of Southern Medical University, Guangzhou, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2018 Accepted 18 June 2018 Available online xxx
Ubiquitin-conjugating enzyme E2S (UBE2S) plays pivotal roles in the progression of human cancers. However, its clinical significance and role in hepatocellular carcinoma (HCC) remain unknown. Here, we show that UBE2S is upregulated in HCC and exhibits oncogenic activities via enhancing the ubiquitination of p53. Increased expression of UBE2S was significantly correlated with higher serum AFP level, higher pathological grade, advanced TNM stage, larger tumor size, vascular invasion and unfavorable patient survivals in two independent cohorts containing a total of 845 patients with HCC. Multivariate analyses by cox regression model suggested UBE2S as an independent factor for overall survival. In vitro experiments demonstrated that UBE2S overexpression promoted, whereas UBE2S knockdown suppressed cell proliferation and migration via modulation of p53 signaling pathway. Ectopic expression of UBE2S upregulated the expression of p53 and its downstream effectors, such as p21 and Cyclin D1. Mechanistically, UBE2S enhanced the ubiquitination of p53 protein to facilitate its degradation in HCC cells. Re-expression of p53 partially attenuated the UBE2S-promoted malignant phenotypes. Collectively, our study provides compelling evidence that UBE2S is a potential prognostic factor and functions as an oncogene in HCC. © 2018 Elsevier Inc. All rights reserved.
Keywords: UBE2S p53 Ubiquitination Hepatocellular carcinoma
1. Introduction Hepatocellular carcinoma (HCC) ranks the fifth most prevalent cancer, and the third as a cause of cancer death worldwide [1]. The morbidity of HCC has been increasing in economically developed regions, including Japan, Western Europe and the United States in recent decades [2], making HCC as a global threat to human lives. In view of that the poor prognosis of HCC, with a median survival time of 4 months, and that the accuracy and reproducibility of markers currently used in clinic to predict survival after surgical resection remain either unsatisfactory or unclear, efforts have been made to uncover the mechanism of HCC progression. Identification of biomarkers with potent prognostic or therapeutic value is of immense
* Corresponding author. E-mail address: [email protected] (S.-G. Su). 1 These authors contributed equally to this work.
importance to develop novel strategies for the clinical management. Ubiquitin-conjugating enzyme E2S (UBE2S, also known as E2EPF), an E2 enzyme [3], coupled with ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C) to elongate K11-linked polyubiquitin chain on substrates for 26 S proteasome-mediated degradation to play pivotal roles in cell division [4e8]. The association of UBE2S and APC/C was inhibited by Emi1 [4], Emi2 [9] and phosphorylation of CDC20 [10]. UBE2S is responsible for the ubiquitin-mediated proteasomal degradation of Sox2 in mouse embryonic stem cells to control the cell differentiation [11]. The critical role of UBE2S in regulating cell cycle, cell differentiation and DNA repair inevitably implicates its involvement of tumorigenesis [12,13]. Aberrant expression of UBE2S has been reported in oral squamous cell carcinoma [14], cervical cancer [15] and renal cancer [16]. High UBE2S expression, co-expressed with the well-known breast cancer markers MKI67 and AURKA, was associated with poor survival in breast cancer [17]. UBE2S mediated the malignant
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Please cite this article in press as: Y.-H. Pan, et al., UBE2S enhances the ubiquitination of p53 and exerts oncogenic activities in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.093
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Y.-H. Pan et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
phenotypes in colorectal cancer via stabilizing b-Catenin through directly interaction with b-Catenin to ubiquitinate its K19 residue via K11 linkage [18]. Knockdown of UBE2S impaired the tumor growth and antagonized the chemo-resistance in glioblastoma [19]. However, its biological function in HCC remain unclear. In this study, the expression of UBE2S and clinical significance were determined in two independent cohorts containing a total of 845 patients with HCC. The role of UBE2S in HCC cell proliferation and migration, as well as the underlying mechanism, was investigated. Our data indicate that UBE2S is upregulated in HCC and exhibits oncogenic activities via enhancing the ubiquitination of p53. 2. Materials and methods 2.1. Patients and specimen A cohort containing 486 paraffin-embedded HCC samples was obtained from May 2005 to December 2012 in Shenzhen People's Hospital (SPH cohort). The mean age of patients was 49.5 years old (ranging from 18 to 79). The median follow-up was 26.5 months. Another cohort consisting of 49 cases with portal vein embolus and 35 fresh specimens was collected. Informed consent was obtained. None of the patients received chemotherapy or radiotherapy before surgery. All samples were anonymous. The use of human samples was approved by the Research Ethics Committee of Second Clinical Medical College of Jinan University and carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). 2.2. Cell lines and transfection HCC cell lines (Bel-7402, Bel-7404 and MHCC-97H) purchased from the Cell Resource Center, Chinese Academy of Science Committee (Shanghai, China) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) in a humidified incubator at 37 C and 5% CO2. The cells were stably transfected with UBE2S overexpression vector or shRNAs with Lipofectamine 2000. Stable cell lines were established by G418 screening for 2 weeks. The shRNA for UBE2S were obtained from Santa Cruz Biotechnology (sc-97109-SH). 2.3. Quantitative real-time PCR Complementary DNA was synthesized from the total RNA, using the PrimeScript RT reagent Kit (TAKARA, USA). qRT-PCR was performed with SYBR Premix ExTaq (TAKARA, USA). The expression of the endogenous 18s was used as control for the normalization of the relative expression of UBE2S. The DCt was calculated. Conditions for RT-PCR was set as the following: 95 C for 10 min, 40 cycles of 94 C for 30 s, 60 C for 30 s, 72 C for 30 s and a final extension of 10 min at 72 C. The primers were as following: UBE2S, forward: 50 - GACCGAAGAACGCAGGAAG-30 and reverse: 50 GTGCGGGGGTAGGTTCTC-3’; b-actin, forward: 50 -TGGCACCCAGCACAATGAA-30 and reverse: 50 -CTAAGTCATAGTCCGCCTAGAAGCA3’.
2.5. Colony formation Stable cells were constructed. Cells were collected and seeded in 6-well plates at a density of 1.0 103 per well and then incubated at 37 C for 10 days. Colonies were fixed with methanol and stained with 0.1% crystal violet and counted. 2.6. Western blot Proteins extracted from HCC fresh tissue or cells with various treatments were fractionated by SDS-PAGE, transferred to PVDF membrane, and then incubated with a primary specific antibody for UBE2S (1:1000, #11878, Cell signaling technology), p53 (1:1000, sc126, Santa-cruz), p21(1:1000, #2947, Cell signaling technology), Cyclin D1 (1:1000, #2978, Cell signaling technology), 14-33s(1:1000, #7413, Cell signaling technology) and b-actin (1:1000, #4970, Cell signaling technology) in 5% of non-fat milk, followed by a horse radish peroxidase (HRP)-conjugated anti-rabbit/mouse second antibody. ECL detection reagent (Amersham Life Science, Piscataway, NJ, USA) was used to visualize the results. 2.7. Immunohistochemistry and scoring TMA sections with a thickness of 4 mm were dewaxed in xylene and graded alcohols, hydrated, and washed in phosphate buffered saline (PBS). After pretreatment in a microwave oven, endogenous peroxidase was inhibited by 3% hydrogen peroxide in methanol for 20 min, followed by avidin-biotin blocking using a biotin-blocking kit (DAKO, Germany). Slides were then incubated with UBE2S (1:500, #11878, Cell signaling technology) or p53 (1:500, sc-126, Santa-cruz), overnight in a moist chamber at 4 C, washed in PBS, and incubated with biotinylated goat anti-rabbit antibody. Slides were developed with the Dako Liquid 3, ’3-diaminobenzidine tetrahydrochloride (DAB) þ Substrate Chromogen System and counterstained with hematoxylin. IHC evaluation was determined by semi-quantitative IHC detection, using the H-score method. The percentage of positivelystained cells was scored as “0” (0%), “1” (1%e25%), “2” (26%e50%), “3” (51%e75%), “4” (76%e100%). Intensity was scored as “0” (negative staining), “1” (weak staining), “2” (moderate staining), and “3” (strong staining). The percentage score was multiplied by the staining intensity score. For each case, 1000 cells were randomly selected and scored. The scores were independently decided by 2 clinical doctors. The median of UBE2S IHC score, which was 4.5, was chosen as the cutoff value to identify high and low expression groups. 2.8. Statistical analysis Continuous variables were expressed as a mean with SEM and analyzed using the Student t-test (2-tailed). Kaplan-Meier analysis (the log-rank test) was used for survival analysis and univariate analysis. The Cox proportional hazards regression model was used to evaluate the independent prognostic value of UBE2S in HCC. The P value less than 0.05 was considered to be statistically significant. 3. Results
2.4. MTT 3.1. UBE2S expression is increased in HCC Stable cells were cultured in 96-well plates for 5 days. 20 ml of MTT (5 mg/ml) was added into the wells for 3 h. The formazan crystals were dissolved in DMSO (150 ml/well). The absorbance at 490 nm of each sample was measured. The cell growth rate was calculated.
To determine the expression of UBE2S in HCC, 35 pairs of HCC fresh tissues were collected. qRT-PCR results showed that UBE2S mRNA was significantly upregulated in HCC tissues, compared with the corresponding nontumorous tissues (Fig. 1A). Consistently, the
Please cite this article in press as: Y.-H. Pan, et al., UBE2S enhances the ubiquitination of p53 and exerts oncogenic activities in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.093
Y.-H. Pan et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8
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Fig. 1. UBE2S expression is increased in HCC. A. The mRNA expression of UBE2S was examined in 35 pairs of fresh HCC specimens by qRT-PCR. B. Proteins from 12 paired HCC tissues were subjected to western blot to determine the protein level of UBE2S. The representative bands and the relative UBE2S protein expressions normalized to b-actin were shown. C. UBE2S protein expression were further evaluated in 486 paraffin-embedded samples and 49 cases with tumor metastasis. The representative images of UBE2S expression in nontumorous (N), primary (T) and metastatic (M) tumors were shown. The relative IHC scores of UBE2S were indicated by box plots. D. The upregulation of UBE2S mRNA was validated in HCC-relative studies from oncomine database. FC: fold change.
protein level of UBE2S in HCC was 3.27-fold higher than that in adjacent liver tissues (Fig. 1B). To further confirm the upregulation of UBE2S, a cohort containing 486 HCC cases was recruited. IHC staining showed that UBE2S was localized in both nucleus and cytoplasm. Compared to the nontumor cells, expression of UBE2S in HCC cells was markedly increased (Fig. 1C). More UBE2S in HCC was
depicted in 80.7% (392/486) of the cases. The expression of UBE2S in portal vein tumor thrombus was further increased, compared with the primary tumor (Fig. 1C). The up-regulation of UBE2S was also validated by the other liver studies from oncomine database (Fig. 1D).
Please cite this article in press as: Y.-H. Pan, et al., UBE2S enhances the ubiquitination of p53 and exerts oncogenic activities in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.093
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3.2. High UBE2S expression is correlated with poor outcomes in HCC
Table 1 The correlation of UBE2S expression and clinical features of patients in SPH cohort. Variable
The clinical significance of UBE2S was next determined. Analysis of The Cancer Genome Atlas (TCGA) data of 359 HCC cases indicated that high expression of UBE2S was significantly associated with higher pathological grade and vascular invasion (Fig. 2AeB). According to the median of UBE2S IHC score, patients in SPH cohort were divided into two groups: low or high UBE2S. High UBE2S expression in HCC was significantly connected with higher serum AFP level (P ¼ 0.037), higher pathological grade (P ¼ 0.034), advanced TNM stage (P ¼ 0.001), larger tumor size (P ¼ 0.002) and vascular invasion (P ¼ 0.009) (Table 1). Kaplan-Meier analyses revealed that patients with high UBE2S expression were frequently accompanied with shorter overall and disease-free survivals in
UBE2S All cases
High expression
Low expression
224 262
115 (51.3%) 128 (48.9%)
109 (48.7%) 134 (51.1%)
441 45
221 (50.1%) 22 (48.9%)
220 (49.9%) 23 (51.1%)
74 412
44 (59.5%) 199 (48.3%)
30 (40.5%) 213 (51.7%)
143 343
82 (57.3%) 161 (46.9%)
61 (42.7%) 182 (53.1%)
412 74
38 (51.4%) 205 (49.8%)
36 (48.6%) 207 (50.2%)
121 365
75 (62.0%) 168 (46.0%)
46 (38.0%) 197 (54.0%)
291 195
153 (52.6%) 90 (46.2%)
138 (47.4%) 105 (53.8%)
57 429
36 (63.2%) 207 (48.3%)
21 (36.8%) 222 (51.7%)
158 328
96 (60.8%) 147 (44.8%)
62 (39.2%) 181 (55.2%)
389 97
206 (53.0%) 37 (38.1%)
183 (47.0%) 60 (61.9%)
295 191
141 (47.8%) 102 (53.4%)
154 (52.2%) 89 (46.6%)
b
Age (years) < 49.5 49.5 Gender Male Female HBV Negative Positive AFP (ng/ml) < 20 20 Cirrhosis Yes No Tumor size (cm) <5 5 Tumor multiplicity Single Multiple Pathological grade G1 G2-G4 TNM I II-IV Vascular invasion Absent Present Tumor Capsule Absent Present
P valuea 0.586
0.876
0.077
0.037
0.801
0.002
0.165
0.034
0.001
0.009
0.227
a
Chi-square test. Median age; AFP, alpha-fetoprotein; HBV, hepatitis B virus; LNM, lymph node metastasis. b
both cohorts (Fig. 2CeD). Stratified survival analyses confirmed the prognostic value of UBE2S in the subgroups of HCC patients (Supplementary Fig. 1). Multivariate analyses by cox regression model suggested UBE2S as an independent factor for overall survival (Hazard ratio ¼ 1.782, 95% confidant interval: 1.465e2.169, P < 0.001) (Table 2). These data indicate that UBE2S serves as a potent prognostic factor in HCC. 3.3. UBE2S promotes cell proliferation and migration in HCC
Fig. 2. High UBE2S expression is correlated with poor outcomes in HCC. A. The correlation between UBE2S expression and the pathological grade of HCC patients in TCGA cohort was determined. B. UBE2S mRNA expression was increased in tumors with micro or macro vascular invasion in TCGA samples. C. The prognostic implication of UBE2S was revealed by Kaplan-Meier analysis in SPH cohort. High expression of UBE2S was correlated with unfavorable overall (left panel) and disease-free (right panel) survivals. D. The prognostic value of UBE2S was confirmed in TCGA cohort.
Gain-of-function and loss-of-function assays were performed to reveal the role of UBE2S in HCC progression. UBE2S was either overexpressed in Bel-7402 and Bel-7404 cells or knocked down in MHCC-97H cells, according to the basal UBE2S expression in HCC cell lines (Fig. 3A and data not shown). MTT assays showed that ectopic expression of UBE2S increased, whereas silence of UBE2S decreased the cell viabilities (Fig. 3B). EdU assays demonstrated that more EdU-positive cells were identified in UBE2S-expressing cells, but cell proliferation was suppressed in cells with UBE2S depletion (Fig. 3C). The effect of UBE2S on cell growth was further validated by colony formation assays. Results showed that UBE2S overexpression enhanced, whereas UBE2S knockdown attenuated the ability of clonogenicity of HCC cells (Fig. 3D). Because UBE2S expression was associated with tumor metastasis in clinical samples, the impact of UBE2S on cell migration was next investigated. Transwell assays presented that UBE2S overexpression resulted in more migrated cells, while UBE2S inhibition suppressed the cell movement (Fig. 3E). Collectively, these findings suggest UBE2S exerts oncogenic activities in HCC.
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Table 2 Univariate and multivariate analyses of UBE2S and overall survival in SPH cohort. Variables
Age (<49.5 vs. 49.5 years) Gender (female vs. male) HBV (positive vs. negative) Liver cirrhosis (yes vs. no) Tumor size (<5 vs. 5 cm) Tumor multiplicity (single vs. multiple) Tumor capsule (absent vs. present) AFP (<20 vs. 20 ng/mL) Vascular invasion (absent vs. present) Pathological grade (G1 vs. G2-G4) TNM (I vs. II-IV) UBE2S expression (high vs. low)
Univariate analysis
Multivariate analysis
HR (95% CI)
P value
0.992 1.047 1.168 1.171 1.764 1.081 1.024 1.683 2.055 1.827 1.293 2.006
0.932 0.780 0.242 0.256 0.000 0.425 0.812 0.000 0.000 0.000 0.011 0.000
(0.822e1.197) (0.759e1.445) (0.900e1.516) (0.892e1.539) (1.404e2.216) (0.892e1.310) (0.845e1.240) (1.358e2.086) (1.632e2.588) (1.347e2.478) (1.060e1.578) (1.653e2.435)
HR (95% CI)
P value
1.485 (1.178e1.872)
0.001
1.371 1.641 1.442 1.032 1.782
0.005 0.000 0.022 0.767 0.000
(1.099e1.711) (1.288e2.092) (1.055e1.971) (0.837e1.273) (1.465e2.169)
AFP, a-fetoprotein; HBsAg, hepatitis B surface antigen; LNM, lymph node metastasis; HR, hazard.
Fig. 3. UBE2S promotes cell proliferation and migration in HCC. A. Cells were transfected with UBE2S overexpression vector or shRNAs for the establishment of stable cell lines. The effects were confirmed by western blot. B. Stable cells were cultured into 96-well plates for 5 days. The cell viabilities were determined by MTT assays. *P < 0.05, **P < 0.01. C. Cells with or without UBE2S were cultured with 300 mg/l of G418 for 10 days. Colonies were counted and indicated. *P < 0.05, **P < 0.01. D. Cells with ectopic UBE2S expression or depletion were stained with EdU. The percentage of EdU-positive cells were counted and shown by histogram. *P < 0.05. E. Transwell assays were performed to evaluate the effect of UBE2S on the cell migration. Representative data and statistical analyses were shown. *P < 0.05, **P < 0.01.
3.4. UBE2S enhances the ubiquitination of p53 in HCC The underlying mechanism of UBE2S-promoted HCC cell growth was next investigated. Gene Set Enrichment Analysis (GSEA) was conducted, using The Cancer Genome Atlas (TCGA) data. Results
showed that p53 signaling pathway was inhibited in HCC cases with high expression of UBE2S mRNA (Fig. 4A), suggesting a role of p53 in UBE2S-mediated phenotypes. In clinical samples, expression of UBE2S was reversely associated with p53. HCC cases with positive UBE2S staining were likely with negative p53 immunoreactivity
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Fig. 4. UBE2S enhances the ubiquitination of p53. A. Gene set enrichment analysis (GSEA) indicated that p53 signaling pathway was suppressed in HCC cases with UBE2S overexpression. B. The correlation of UBE2S and p53 in 486 HCC tissues were determined. Representative IHC pictures and the statistical analysis were shown. C. Proteins from stable cells were subjected to western blot to examine the expression of genes involved in p53 pathway, including p53, p21, Noxa and CyclinD1. D. Using cycloheximide (CHX, 20 mg/ ml), the protein stability of p53 in UBE2S-expressing cells was determined by western blot. The relative protein densities were calculated and shown by curves. Data are mean ± SEM of three independent experiments. E. Cells were transfected with HA-Ub and/or UBE2S for 36 h. The ubiquitination of p53 was determined. F,G,H. The effect of p53 reexpression on the UBE2S-promoted malignant phenotypes, using colony formation (F), EdU staining (G), and Transwell assay (H). *P < 0.05.
(Fig. 4B). In HCC cells, the expression of p53, as well as its downstream effectors including p21, Cyclin D1, Noxa and 14-3-3s, was down-regulated by UBE2S overexpression, but up-regulated by UBE2S knockdown (Fig. 4C). Since the transcript of p53 was not affected by UBE2S (Supplementary Fig. 2), the post-transcriptional regulation of p53 by UBE2S was determined. Using cycloheximide (CHX, 20 mg/ml), the protein stability of p53 was determined by
western blot. The expression of p53 was decreased from 53.6% in control Bel-7402 cells to 33.5% in UBE2S-expressing cells, following the CHX incubation for 15 min (Fig. 4D). Further studies demonstrated that the ubiquitination of p53 was enhanced by the overexpression of UBE2S in both Bel-7402 and Bel-7404 cells (Fig. 4E). In order to determine the role of p53 in UBE2S-mediated phenotypes, rescue experiments were performed. Overexpression of
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p53 in UBE2S-expressing HCC cells markedly attenuated the cell proliferation, as shown by the colony formation and EdU assays (Fig. 4FeG). On the other hand, the cell migration induced by UBE2S was mainly inhibited by the ectopic p53 expression (Fig. 4H). These findings suggest that UBE2S facilitates the p53 ubiquitination to function as an oncogene in HCC.
Appendix A. Supplementary data
4. Discussion
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Fountzilas, Prognostic significance of UBE2C mRNA expression in high-risk early breast cancer. A hellenic cooperative oncology group (HeCOG) study, Ann. Oncol. 23 (2012) 1422e1427. [22] M.T. Berlingieri, P. Pallante, M. Guida, C. Nappi, V. Masciullo, G. Scambia, A. Ferraro, V. Leone, A. Sboner, M. Barbareschi, A. Ferro, G. Troncone, A. Fusco, UbcH10 expression may be a useful tool in the prognosis of ovarian
The high morbidity and mortality of HCC makes it a global threat of human lives. Despite new strategies have been applied to the diagnosis and treatment of HCC, most of the patients died within 3 years after HCC was diagnosed [20]. Progressions in molecular biology have recently led to the rapid development of individualized cancer management. Here, we identify UBE2S as an independent prognostic factor for HCC and an oncogene through enhancing the ubiquitin-mediated proteasomal degradation of p53 protein. Ubiquitination process is very important for the cellular homeostasis and is controlled by the sequential actions of three enzymes: an ubiquitin-activating enzyme (E1), an ubiquitinconjugating enzyme (E2) and an ubiquitin ligase (E3) [3]. There are approximately 40 members of the E2 family. Some of them have been reported to be of prognostic value in human cancers. For example, high UBE2C expression predicted poor patient survivals in breast cancer [21] and ovarian carcinoma [22]. UBE2T expression was correlated with clinical outcomes in patients with gastric cancer [23] or HCC [24]. In this study, elevated expression of UBE2S was connected with unfavorable overall and disease-free survivals in two independent cohorts consisting of 845 patients with HCC. This may be due to the fact that patients with high expression of UBE2S were usually accompanied with higher pathological grade and tumor vascular invasion. The prognostic implication of UBE2S was also confirmed in Glioma [25] and breast cancer [26]. These data suggest E2 proteins are of clinical significance in post-surgery surveillance of cancer patients. Literatures reported that UBE2S co-worked with APC/C to catalyze polyubiquitination of key regulators, such as Cyclin B1 and CDC20 [27], to control the cell division that triggers the cell proliferation. Our data presented that UBE2S was capable of promoting cell growth and migration. GSEA analysis indicated p53 signaling pathway was suppressed in HCC cases with high expression of UBE2S. Further studies revealed that UBE2S downregulated p53, another key cell cycle regulator, and its downstream effectors, such as p21 and Cyclin D1, via enhancing the ubiquitination of p53 for protein degradation in HCC cells. In a study of UBE2S in oral squamous cell carcinoma, p21 induction was depicted in cells with UBE2S knockdown [14]. However, no protein-protein interaction was found between UBE2S and p53 (data not shown). As a result, the detailed mechanism of UBE2S-mediated p53 degradation requires further investigation. In our previous study, UBE2T was also identified as an upstream regulator of p53 [24]. Again, no direct binding was observed between UBE2T and p53. Whether UBE2S coworks with UBE2T and/or other ubiquitin-proteasome proteins to facilitate the downregulation of p53 in HCC cells remain so far unclear. Results of rescue experiments showed that re-expression of p53 partially attenuated the UBE2S-promoted cell proliferation and migration. Collectively, these data suggest UBE2S serves a promising prognostic factor in HCC and exerts pro-HCC activities via suppression of p53 pathway. The newly identified UBE2S/p53 axis may represent a new potential therapeutic target for HCC intervention. Conflicts of interest Authors declare that there is no conflict of interest.
Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.06.093. References
Please cite this article in press as: Y.-H. Pan, et al., UBE2S enhances the ubiquitination of p53 and exerts oncogenic activities in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.093
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carcinomas, Oncogene 26 (2007) 2136e2140. [23] C. Luo, Y. Yao, Z. Yu, H. Zhou, L. Guo, J. Zhang, H. Cao, G. Zhang, Y. Li, Z. Jiao, UBE2T knockdown inhibits gastric cancer progression, Oncotarget 8 (2017) 32639e32654. [24] L.P. Liu, M. Yang, Q.Z. Peng, M.Y. Li, Y.S. Zhang, Y.H. Guo, Y. Chen, S.Y. Bao, UBE2T promotes hepatocellular carcinoma cell growth via ubiquitination of p53, Biochem. Biophys. Res. Commun. 493 (2017) 20e27. [25] X. Zhang, F. Zhao, S. Zhang, Y. Song, Ectopic high expression of E2-EPF ubiquitin Carrier protein indicates a more unfavorable prognosis in brain glioma,
Genet. Test. Mol. Biomarkers 21 (2017) 242e247. [26] C. Clarke, S.F. Madden, P. Doolan, S.T. Aherne, H. Joyce, L. O'Driscoll, W.M. Gallagher, B.T. Hennessy, M. Moriarty, J. Crown, S. Kennedy, M. Clynes, Correlating transcriptional networks to breast cancer survival: a large-scale coexpression analysis, Carcinogenesis 34 (2013) 2300e2308. [27] N.V. Dimova, N.A. Hathaway, B.H. Lee, D.S. Kirkpatrick, M.L. Berkowitz, S.P. Gygi, D. Finley, R.W. King, APC/C-mediated multiple monoubiquitylation provides an alternative degradation signal for cyclin B1, Nat. Cell Biol. 14 (2012) 168e176.
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UBE2S enhances the ubiquitination of p53 and exerts oncogenic activities in hepatocellular carcinoma Ying-Hua Pan a, 1, Mei Yang b, 1, Li-ping Liu c, Dan-Chun Wu a, Ming-yue Li c, Shu-Guang Su d, * a
Department of Rheumatology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China Department of Gastroenterology, Dongguan Third People's Hospital, Dongguan, China Department of Hepatobiliary and Pancreatic Surgery, Shenzhen People's Hospital, Second Clinical Medical College of Jinan University, Shenzhen, Guangdong Province, China d Department of Pathology, The Affiliated Hexian Memorial Hospital of Southern Medical University, Guangzhou, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2018 Accepted 18 June 2018 Available online xxx
Ubiquitin-conjugating enzyme E2S (UBE2S) plays pivotal roles in the progression of human cancers. However, its clinical significance and role in hepatocellular carcinoma (HCC) remain unknown. Here, we show that UBE2S is upregulated in HCC and exhibits oncogenic activities via enhancing the ubiquitination of p53. Increased expression of UBE2S was significantly correlated with higher serum AFP level, higher pathological grade, advanced TNM stage, larger tumor size, vascular invasion and unfavorable patient survivals in two independent cohorts containing a total of 845 patients with HCC. Multivariate analyses by cox regression model suggested UBE2S as an independent factor for overall survival. In vitro experiments demonstrated that UBE2S overexpression promoted, whereas UBE2S knockdown suppressed cell proliferation and migration via modulation of p53 signaling pathway. Ectopic expression of UBE2S upregulated the expression of p53 and its downstream effectors, such as p21 and Cyclin D1. Mechanistically, UBE2S enhanced the ubiquitination of p53 protein to facilitate its degradation in HCC cells. Re-expression of p53 partially attenuated the UBE2S-promoted malignant phenotypes. Collectively, our study provides compelling evidence that UBE2S is a potential prognostic factor and functions as an oncogene in HCC. © 2018 Elsevier Inc. All rights reserved.
Keywords: UBE2S p53 Ubiquitination Hepatocellular carcinoma
1. Introduction Hepatocellular carcinoma (HCC) ranks the fifth most prevalent cancer, and the third as a cause of cancer death worldwide [1]. The morbidity of HCC has been increasing in economically developed regions, including Japan, Western Europe and the United States in recent decades [2], making HCC as a global threat to human lives. In view of that the poor prognosis of HCC, with a median survival time of 4 months, and that the accuracy and reproducibility of markers currently used in clinic to predict survival after surgical resection remain either unsatisfactory or unclear, efforts have been made to uncover the mechanism of HCC progression. Identification of biomarkers with potent prognostic or therapeutic value is of immense
* Corresponding author. E-mail address: [email protected] (S.-G. Su). 1 These authors contributed equally to this work.
importance to develop novel strategies for the clinical management. Ubiquitin-conjugating enzyme E2S (UBE2S, also known as E2EPF), an E2 enzyme [3], coupled with ubiquitin ligase anaphasepromoting complex/cyclosome (APC/C) to elongate K11-linked polyubiquitin chain on substrates for 26 S proteasome-mediated degradation to play pivotal roles in cell division [4e8]. The association of UBE2S and APC/C was inhibited by Emi1 [4], Emi2 [9] and phosphorylation of CDC20 [10]. UBE2S is responsible for the ubiquitin-mediated proteasomal degradation of Sox2 in mouse embryonic stem cells to control the cell differentiation [11]. The critical role of UBE2S in regulating cell cycle, cell differentiation and DNA repair inevitably implicates its involvement of tumorigenesis [12,13]. Aberrant expression of UBE2S has been reported in oral squamous cell carcinoma [14], cervical cancer [15] and renal cancer [16]. High UBE2S expression, co-expressed with the well-known breast cancer markers MKI67 and AURKA, was associated with poor survival in breast cancer [17]. UBE2S mediated the malignant
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phenotypes in colorectal cancer via stabilizing b-Catenin through directly interaction with b-Catenin to ubiquitinate its K19 residue via K11 linkage [18]. Knockdown of UBE2S impaired the tumor growth and antagonized the chemo-resistance in glioblastoma [19]. However, its biological function in HCC remain unclear. In this study, the expression of UBE2S and clinical significance were determined in two independent cohorts containing a total of 845 patients with HCC. The role of UBE2S in HCC cell proliferation and migration, as well as the underlying mechanism, was investigated. Our data indicate that UBE2S is upregulated in HCC and exhibits oncogenic activities via enhancing the ubiquitination of p53. 2. Materials and methods 2.1. Patients and specimen A cohort containing 486 paraffin-embedded HCC samples was obtained from May 2005 to December 2012 in Shenzhen People's Hospital (SPH cohort). The mean age of patients was 49.5 years old (ranging from 18 to 79). The median follow-up was 26.5 months. Another cohort consisting of 49 cases with portal vein embolus and 35 fresh specimens was collected. Informed consent was obtained. None of the patients received chemotherapy or radiotherapy before surgery. All samples were anonymous. The use of human samples was approved by the Research Ethics Committee of Second Clinical Medical College of Jinan University and carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). 2.2. Cell lines and transfection HCC cell lines (Bel-7402, Bel-7404 and MHCC-97H) purchased from the Cell Resource Center, Chinese Academy of Science Committee (Shanghai, China) were maintained in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) in a humidified incubator at 37 C and 5% CO2. The cells were stably transfected with UBE2S overexpression vector or shRNAs with Lipofectamine 2000. Stable cell lines were established by G418 screening for 2 weeks. The shRNA for UBE2S were obtained from Santa Cruz Biotechnology (sc-97109-SH). 2.3. Quantitative real-time PCR Complementary DNA was synthesized from the total RNA, using the PrimeScript RT reagent Kit (TAKARA, USA). qRT-PCR was performed with SYBR Premix ExTaq (TAKARA, USA). The expression of the endogenous 18s was used as control for the normalization of the relative expression of UBE2S. The DCt was calculated. Conditions for RT-PCR was set as the following: 95 C for 10 min, 40 cycles of 94 C for 30 s, 60 C for 30 s, 72 C for 30 s and a final extension of 10 min at 72 C. The primers were as following: UBE2S, forward: 50 - GACCGAAGAACGCAGGAAG-30 and reverse: 50 GTGCGGGGGTAGGTTCTC-3’; b-actin, forward: 50 -TGGCACCCAGCACAATGAA-30 and reverse: 50 -CTAAGTCATAGTCCGCCTAGAAGCA3’.
2.5. Colony formation Stable cells were constructed. Cells were collected and seeded in 6-well plates at a density of 1.0 103 per well and then incubated at 37 C for 10 days. Colonies were fixed with methanol and stained with 0.1% crystal violet and counted. 2.6. Western blot Proteins extracted from HCC fresh tissue or cells with various treatments were fractionated by SDS-PAGE, transferred to PVDF membrane, and then incubated with a primary specific antibody for UBE2S (1:1000, #11878, Cell signaling technology), p53 (1:1000, sc126, Santa-cruz), p21(1:1000, #2947, Cell signaling technology), Cyclin D1 (1:1000, #2978, Cell signaling technology), 14-33s(1:1000, #7413, Cell signaling technology) and b-actin (1:1000, #4970, Cell signaling technology) in 5% of non-fat milk, followed by a horse radish peroxidase (HRP)-conjugated anti-rabbit/mouse second antibody. ECL detection reagent (Amersham Life Science, Piscataway, NJ, USA) was used to visualize the results. 2.7. Immunohistochemistry and scoring TMA sections with a thickness of 4 mm were dewaxed in xylene and graded alcohols, hydrated, and washed in phosphate buffered saline (PBS). After pretreatment in a microwave oven, endogenous peroxidase was inhibited by 3% hydrogen peroxide in methanol for 20 min, followed by avidin-biotin blocking using a biotin-blocking kit (DAKO, Germany). Slides were then incubated with UBE2S (1:500, #11878, Cell signaling technology) or p53 (1:500, sc-126, Santa-cruz), overnight in a moist chamber at 4 C, washed in PBS, and incubated with biotinylated goat anti-rabbit antibody. Slides were developed with the Dako Liquid 3, ’3-diaminobenzidine tetrahydrochloride (DAB) þ Substrate Chromogen System and counterstained with hematoxylin. IHC evaluation was determined by semi-quantitative IHC detection, using the H-score method. The percentage of positivelystained cells was scored as “0” (0%), “1” (1%e25%), “2” (26%e50%), “3” (51%e75%), “4” (76%e100%). Intensity was scored as “0” (negative staining), “1” (weak staining), “2” (moderate staining), and “3” (strong staining). The percentage score was multiplied by the staining intensity score. For each case, 1000 cells were randomly selected and scored. The scores were independently decided by 2 clinical doctors. The median of UBE2S IHC score, which was 4.5, was chosen as the cutoff value to identify high and low expression groups. 2.8. Statistical analysis Continuous variables were expressed as a mean with SEM and analyzed using the Student t-test (2-tailed). Kaplan-Meier analysis (the log-rank test) was used for survival analysis and univariate analysis. The Cox proportional hazards regression model was used to evaluate the independent prognostic value of UBE2S in HCC. The P value less than 0.05 was considered to be statistically significant. 3. Results
2.4. MTT 3.1. UBE2S expression is increased in HCC Stable cells were cultured in 96-well plates for 5 days. 20 ml of MTT (5 mg/ml) was added into the wells for 3 h. The formazan crystals were dissolved in DMSO (150 ml/well). The absorbance at 490 nm of each sample was measured. The cell growth rate was calculated.
To determine the expression of UBE2S in HCC, 35 pairs of HCC fresh tissues were collected. qRT-PCR results showed that UBE2S mRNA was significantly upregulated in HCC tissues, compared with the corresponding nontumorous tissues (Fig. 1A). Consistently, the
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Fig. 1. UBE2S expression is increased in HCC. A. The mRNA expression of UBE2S was examined in 35 pairs of fresh HCC specimens by qRT-PCR. B. Proteins from 12 paired HCC tissues were subjected to western blot to determine the protein level of UBE2S. The representative bands and the relative UBE2S protein expressions normalized to b-actin were shown. C. UBE2S protein expression were further evaluated in 486 paraffin-embedded samples and 49 cases with tumor metastasis. The representative images of UBE2S expression in nontumorous (N), primary (T) and metastatic (M) tumors were shown. The relative IHC scores of UBE2S were indicated by box plots. D. The upregulation of UBE2S mRNA was validated in HCC-relative studies from oncomine database. FC: fold change.
protein level of UBE2S in HCC was 3.27-fold higher than that in adjacent liver tissues (Fig. 1B). To further confirm the upregulation of UBE2S, a cohort containing 486 HCC cases was recruited. IHC staining showed that UBE2S was localized in both nucleus and cytoplasm. Compared to the nontumor cells, expression of UBE2S in HCC cells was markedly increased (Fig. 1C). More UBE2S in HCC was
depicted in 80.7% (392/486) of the cases. The expression of UBE2S in portal vein tumor thrombus was further increased, compared with the primary tumor (Fig. 1C). The up-regulation of UBE2S was also validated by the other liver studies from oncomine database (Fig. 1D).
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3.2. High UBE2S expression is correlated with poor outcomes in HCC
Table 1 The correlation of UBE2S expression and clinical features of patients in SPH cohort. Variable
The clinical significance of UBE2S was next determined. Analysis of The Cancer Genome Atlas (TCGA) data of 359 HCC cases indicated that high expression of UBE2S was significantly associated with higher pathological grade and vascular invasion (Fig. 2AeB). According to the median of UBE2S IHC score, patients in SPH cohort were divided into two groups: low or high UBE2S. High UBE2S expression in HCC was significantly connected with higher serum AFP level (P ¼ 0.037), higher pathological grade (P ¼ 0.034), advanced TNM stage (P ¼ 0.001), larger tumor size (P ¼ 0.002) and vascular invasion (P ¼ 0.009) (Table 1). Kaplan-Meier analyses revealed that patients with high UBE2S expression were frequently accompanied with shorter overall and disease-free survivals in
UBE2S All cases
High expression
Low expression
224 262
115 (51.3%) 128 (48.9%)
109 (48.7%) 134 (51.1%)
441 45
221 (50.1%) 22 (48.9%)
220 (49.9%) 23 (51.1%)
74 412
44 (59.5%) 199 (48.3%)
30 (40.5%) 213 (51.7%)
143 343
82 (57.3%) 161 (46.9%)
61 (42.7%) 182 (53.1%)
412 74
38 (51.4%) 205 (49.8%)
36 (48.6%) 207 (50.2%)
121 365
75 (62.0%) 168 (46.0%)
46 (38.0%) 197 (54.0%)
291 195
153 (52.6%) 90 (46.2%)
138 (47.4%) 105 (53.8%)
57 429
36 (63.2%) 207 (48.3%)
21 (36.8%) 222 (51.7%)
158 328
96 (60.8%) 147 (44.8%)
62 (39.2%) 181 (55.2%)
389 97
206 (53.0%) 37 (38.1%)
183 (47.0%) 60 (61.9%)
295 191
141 (47.8%) 102 (53.4%)
154 (52.2%) 89 (46.6%)
b
Age (years) < 49.5 49.5 Gender Male Female HBV Negative Positive AFP (ng/ml) < 20 20 Cirrhosis Yes No Tumor size (cm) <5 5 Tumor multiplicity Single Multiple Pathological grade G1 G2-G4 TNM I II-IV Vascular invasion Absent Present Tumor Capsule Absent Present
P valuea 0.586
0.876
0.077
0.037
0.801
0.002
0.165
0.034
0.001
0.009
0.227
a
Chi-square test. Median age; AFP, alpha-fetoprotein; HBV, hepatitis B virus; LNM, lymph node metastasis. b
both cohorts (Fig. 2CeD). Stratified survival analyses confirmed the prognostic value of UBE2S in the subgroups of HCC patients (Supplementary Fig. 1). Multivariate analyses by cox regression model suggested UBE2S as an independent factor for overall survival (Hazard ratio ¼ 1.782, 95% confidant interval: 1.465e2.169, P < 0.001) (Table 2). These data indicate that UBE2S serves as a potent prognostic factor in HCC. 3.3. UBE2S promotes cell proliferation and migration in HCC
Fig. 2. High UBE2S expression is correlated with poor outcomes in HCC. A. The correlation between UBE2S expression and the pathological grade of HCC patients in TCGA cohort was determined. B. UBE2S mRNA expression was increased in tumors with micro or macro vascular invasion in TCGA samples. C. The prognostic implication of UBE2S was revealed by Kaplan-Meier analysis in SPH cohort. High expression of UBE2S was correlated with unfavorable overall (left panel) and disease-free (right panel) survivals. D. The prognostic value of UBE2S was confirmed in TCGA cohort.
Gain-of-function and loss-of-function assays were performed to reveal the role of UBE2S in HCC progression. UBE2S was either overexpressed in Bel-7402 and Bel-7404 cells or knocked down in MHCC-97H cells, according to the basal UBE2S expression in HCC cell lines (Fig. 3A and data not shown). MTT assays showed that ectopic expression of UBE2S increased, whereas silence of UBE2S decreased the cell viabilities (Fig. 3B). EdU assays demonstrated that more EdU-positive cells were identified in UBE2S-expressing cells, but cell proliferation was suppressed in cells with UBE2S depletion (Fig. 3C). The effect of UBE2S on cell growth was further validated by colony formation assays. Results showed that UBE2S overexpression enhanced, whereas UBE2S knockdown attenuated the ability of clonogenicity of HCC cells (Fig. 3D). Because UBE2S expression was associated with tumor metastasis in clinical samples, the impact of UBE2S on cell migration was next investigated. Transwell assays presented that UBE2S overexpression resulted in more migrated cells, while UBE2S inhibition suppressed the cell movement (Fig. 3E). Collectively, these findings suggest UBE2S exerts oncogenic activities in HCC.
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Table 2 Univariate and multivariate analyses of UBE2S and overall survival in SPH cohort. Variables
Age (<49.5 vs. 49.5 years) Gender (female vs. male) HBV (positive vs. negative) Liver cirrhosis (yes vs. no) Tumor size (<5 vs. 5 cm) Tumor multiplicity (single vs. multiple) Tumor capsule (absent vs. present) AFP (<20 vs. 20 ng/mL) Vascular invasion (absent vs. present) Pathological grade (G1 vs. G2-G4) TNM (I vs. II-IV) UBE2S expression (high vs. low)
Univariate analysis
Multivariate analysis
HR (95% CI)
P value
0.992 1.047 1.168 1.171 1.764 1.081 1.024 1.683 2.055 1.827 1.293 2.006
0.932 0.780 0.242 0.256 0.000 0.425 0.812 0.000 0.000 0.000 0.011 0.000
(0.822e1.197) (0.759e1.445) (0.900e1.516) (0.892e1.539) (1.404e2.216) (0.892e1.310) (0.845e1.240) (1.358e2.086) (1.632e2.588) (1.347e2.478) (1.060e1.578) (1.653e2.435)
HR (95% CI)
P value
1.485 (1.178e1.872)
0.001
1.371 1.641 1.442 1.032 1.782
0.005 0.000 0.022 0.767 0.000
(1.099e1.711) (1.288e2.092) (1.055e1.971) (0.837e1.273) (1.465e2.169)
AFP, a-fetoprotein; HBsAg, hepatitis B surface antigen; LNM, lymph node metastasis; HR, hazard.
Fig. 3. UBE2S promotes cell proliferation and migration in HCC. A. Cells were transfected with UBE2S overexpression vector or shRNAs for the establishment of stable cell lines. The effects were confirmed by western blot. B. Stable cells were cultured into 96-well plates for 5 days. The cell viabilities were determined by MTT assays. *P < 0.05, **P < 0.01. C. Cells with or without UBE2S were cultured with 300 mg/l of G418 for 10 days. Colonies were counted and indicated. *P < 0.05, **P < 0.01. D. Cells with ectopic UBE2S expression or depletion were stained with EdU. The percentage of EdU-positive cells were counted and shown by histogram. *P < 0.05. E. Transwell assays were performed to evaluate the effect of UBE2S on the cell migration. Representative data and statistical analyses were shown. *P < 0.05, **P < 0.01.
3.4. UBE2S enhances the ubiquitination of p53 in HCC The underlying mechanism of UBE2S-promoted HCC cell growth was next investigated. Gene Set Enrichment Analysis (GSEA) was conducted, using The Cancer Genome Atlas (TCGA) data. Results
showed that p53 signaling pathway was inhibited in HCC cases with high expression of UBE2S mRNA (Fig. 4A), suggesting a role of p53 in UBE2S-mediated phenotypes. In clinical samples, expression of UBE2S was reversely associated with p53. HCC cases with positive UBE2S staining were likely with negative p53 immunoreactivity
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Fig. 4. UBE2S enhances the ubiquitination of p53. A. Gene set enrichment analysis (GSEA) indicated that p53 signaling pathway was suppressed in HCC cases with UBE2S overexpression. B. The correlation of UBE2S and p53 in 486 HCC tissues were determined. Representative IHC pictures and the statistical analysis were shown. C. Proteins from stable cells were subjected to western blot to examine the expression of genes involved in p53 pathway, including p53, p21, Noxa and CyclinD1. D. Using cycloheximide (CHX, 20 mg/ ml), the protein stability of p53 in UBE2S-expressing cells was determined by western blot. The relative protein densities were calculated and shown by curves. Data are mean ± SEM of three independent experiments. E. Cells were transfected with HA-Ub and/or UBE2S for 36 h. The ubiquitination of p53 was determined. F,G,H. The effect of p53 reexpression on the UBE2S-promoted malignant phenotypes, using colony formation (F), EdU staining (G), and Transwell assay (H). *P < 0.05.
(Fig. 4B). In HCC cells, the expression of p53, as well as its downstream effectors including p21, Cyclin D1, Noxa and 14-3-3s, was down-regulated by UBE2S overexpression, but up-regulated by UBE2S knockdown (Fig. 4C). Since the transcript of p53 was not affected by UBE2S (Supplementary Fig. 2), the post-transcriptional regulation of p53 by UBE2S was determined. Using cycloheximide (CHX, 20 mg/ml), the protein stability of p53 was determined by
western blot. The expression of p53 was decreased from 53.6% in control Bel-7402 cells to 33.5% in UBE2S-expressing cells, following the CHX incubation for 15 min (Fig. 4D). Further studies demonstrated that the ubiquitination of p53 was enhanced by the overexpression of UBE2S in both Bel-7402 and Bel-7404 cells (Fig. 4E). In order to determine the role of p53 in UBE2S-mediated phenotypes, rescue experiments were performed. Overexpression of
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p53 in UBE2S-expressing HCC cells markedly attenuated the cell proliferation, as shown by the colony formation and EdU assays (Fig. 4FeG). On the other hand, the cell migration induced by UBE2S was mainly inhibited by the ectopic p53 expression (Fig. 4H). These findings suggest that UBE2S facilitates the p53 ubiquitination to function as an oncogene in HCC.
Appendix A. Supplementary data
4. Discussion
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The high morbidity and mortality of HCC makes it a global threat of human lives. Despite new strategies have been applied to the diagnosis and treatment of HCC, most of the patients died within 3 years after HCC was diagnosed [20]. Progressions in molecular biology have recently led to the rapid development of individualized cancer management. Here, we identify UBE2S as an independent prognostic factor for HCC and an oncogene through enhancing the ubiquitin-mediated proteasomal degradation of p53 protein. Ubiquitination process is very important for the cellular homeostasis and is controlled by the sequential actions of three enzymes: an ubiquitin-activating enzyme (E1), an ubiquitinconjugating enzyme (E2) and an ubiquitin ligase (E3) [3]. There are approximately 40 members of the E2 family. Some of them have been reported to be of prognostic value in human cancers. For example, high UBE2C expression predicted poor patient survivals in breast cancer [21] and ovarian carcinoma [22]. UBE2T expression was correlated with clinical outcomes in patients with gastric cancer [23] or HCC [24]. In this study, elevated expression of UBE2S was connected with unfavorable overall and disease-free survivals in two independent cohorts consisting of 845 patients with HCC. This may be due to the fact that patients with high expression of UBE2S were usually accompanied with higher pathological grade and tumor vascular invasion. The prognostic implication of UBE2S was also confirmed in Glioma [25] and breast cancer [26]. These data suggest E2 proteins are of clinical significance in post-surgery surveillance of cancer patients. Literatures reported that UBE2S co-worked with APC/C to catalyze polyubiquitination of key regulators, such as Cyclin B1 and CDC20 [27], to control the cell division that triggers the cell proliferation. Our data presented that UBE2S was capable of promoting cell growth and migration. GSEA analysis indicated p53 signaling pathway was suppressed in HCC cases with high expression of UBE2S. Further studies revealed that UBE2S downregulated p53, another key cell cycle regulator, and its downstream effectors, such as p21 and Cyclin D1, via enhancing the ubiquitination of p53 for protein degradation in HCC cells. In a study of UBE2S in oral squamous cell carcinoma, p21 induction was depicted in cells with UBE2S knockdown [14]. However, no protein-protein interaction was found between UBE2S and p53 (data not shown). As a result, the detailed mechanism of UBE2S-mediated p53 degradation requires further investigation. In our previous study, UBE2T was also identified as an upstream regulator of p53 [24]. Again, no direct binding was observed between UBE2T and p53. Whether UBE2S coworks with UBE2T and/or other ubiquitin-proteasome proteins to facilitate the downregulation of p53 in HCC cells remain so far unclear. Results of rescue experiments showed that re-expression of p53 partially attenuated the UBE2S-promoted cell proliferation and migration. Collectively, these data suggest UBE2S serves a promising prognostic factor in HCC and exerts pro-HCC activities via suppression of p53 pathway. The newly identified UBE2S/p53 axis may represent a new potential therapeutic target for HCC intervention. Conflicts of interest Authors declare that there is no conflict of interest.
Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.06.093. References
Please cite this article in press as: Y.-H. Pan, et al., UBE2S enhances the ubiquitination of p53 and exerts oncogenic activities in hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.06.093
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