Cancer Biol Med 2020. doi: 10.20892/j.issn.2095-3941.2019.0279

ORIGINAL ARTICLE

Identification of a potential tumor suppressor , UBL3, in non-small cell lung cancer

Xinchun Zhao1,2,3, Yongchun Zhou4, Qian Hu2,5, Sanhui Gao2,3, Jie Liu2,3, Hong Yu2,5, Yanfei Zhang2,3, Guizhen Wang2,3, Yunchao Huang4, Guangbiao Zhou2,3 1School of Life Sciences, University of Science and Technology of China, Hefei 230026, China; 2State Key Laboratory of Molecular Oncology, National Cancer Center, National Clinical Research Center for Cancer, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China; 3State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; 4Department of Thoracic Surgery, the Third Affiliated Hospital of Kunming Medical University, Kunming 650106, China;5 School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China

ABSTRACT Objective: Oncogenes have been shown to be drivers of non-small cell lung cancer (NSCLC), yet the tumor suppressing involved in lung carcinogenesis remain to be systematically investigated. This study aimed to identify tumor suppressing pathway genes (UPGs) that were critical to lung tumorigenesis. Methods: The 696 UPGs were silenced by an siRNA screening in NSCLC cells; the potential tumor suppressing UPGs were analyzed, and their clinical significance was investigated. Results: We reported that silencing of 11 UPGs resulted in enhanced proliferation of NSCLC cells, and four UPGs (UBL3, TRIM22, UBE2G2, and MARCH1) were significantly downregulated in tumor samples compared to that in normal lung tissues and their expression levels were positively associated with overall survival (OS) of NSCLC patients. Among these genes, UBL3 was the most significant one. UBL3 expression was decreased in tumor samples compared to that in paired normal lung tissues in 59/86 (68.6%) NSCLCs, was correlated with TNM stage and sex of NSCLC patients, and was significantly higher in non-smoking patients than in smoking patients. Silencing UBL3 accelerated cell proliferation and ectopic expression of UBL3 suppressed NSCLC in vitro and in vivo. Conclusions: These results showed that UBL3 represented a tumor suppressor in NSCLC and may have potential for use in therapeutics and for the prediction of clinical outcome of patients. KEYWORDS Lung cancer; ubiquitin pathway genes; UBL3; tumor suppressor; prognosis

Introduction lung cancer is a disease of the genome3-5. Abnormalities in oncogenes and tumor suppressors act as drivers to initiate the Lung cancer is the most common cause of cancer-related onset and progression of NSCLCs. However, druggable driver mortality, which kills 1.8 million people worldwide each molecules are found in only a minority of patients; hence, year1. Lung cancer comprises small cell lung cancer (SCLC) efforts should be made to uncover the driver genes in the 2 and non-small cell lung cancer (NSCLC). NSCLC divides majority of lung cancers . into large cell carcinoma, lung adenocarcinoma (LUAD), The ubiquitin (Ub)-dependent modification system is and lung squamous cell carcinoma (LUSC)2. Comprehensive one of the degradation systems within the cell and is sequencing efforts over the past decade have confirmed that involved in a variety of cellular processes. In this process, Ub is activated by a Ub-activating enzyme, E1, and transferred

Correspondence to: Guangbiao Zhou to a Ub-conjugating enzyme, E2, which attaches Ub to a tar- E-mail: [email protected] get protein through the ε-amino group of a lysine residue ORCID ID: https://orcid.org/0000-0002-6327-2316 with the help of a Ub-protein ligase, E3. The ubiquitinated Received August 11, 2019; accepted October 24, 2019. target protein is then recognized and degraded by the 26S Available at www.cancerbiomed.org 6 ©2020 Cancer Biology & Medicine. Creative Commons proteasome . Ub-like (UBLs) usually have a UBL Attribution-NonCommercial 4.0 International License domain and have been reported to act as post-translational Cancer Biol Med Vol 17, No 1 February 2020 77 modifiers. The modification of proteins by UBLs proceeds Dharmacon (Lafayette, CO, USA). The small interfering RNAs through an enzyme cascade similar to that used by Ub. As (siRNAs) were transfected into cells using DharmFECT trans- most UBLs have far fewer known substrates than are known fection reagent (#T-2001-01), and cell viability was assessed by for Ub, they require a more limited number of E2-type conju- CellTiter-Glo Reagent (Promega, Fitchburg, WI, USA) accord- gating enzymes and E3-type ligases7. Small Ub-like modifier ing to the manufacturer’s instructions. Each SMARTpool (SUMO)8 and neuronal precursor cell-expressed develop- experiment was performed in duplicate. The Z-score was cal- mentally downregulated protein-8 (Nedd8)9 are two well- culated as follows: z = (x − m)/s, where x is the raw score to be known UBLs and have been reported to play important roles standardized, m is the mean of the plate, and s is the standard in cancer. Both SUMO and Nedd8 are two well-studied UBLs: deviation (SD) of the plate13. The Z-score of a gene reflects its SUMO is involved in several cellular activities, including cell requirement for cell proliferation, and a Z-score of ≤ -2 indi- cycle control, nuclear transport and responses to viral infec- cates that the gene is required for cell proliferation; silencing tions, and Nedd8 functions in the regulation of E3 ubiqui- of the gene significantly inhibits cell proliferation. However, a tin-protein ligases. These two UBLs are implicated in tum- Z score of ≥ 2 indicates that the gene inhibits lung cancer cell origenesis8,9. Some oncogenic Ub-pathway genes (UPGs) proliferation and that inhibition of the gene significantly pro- have been reported in NSCLCs10-12, but the roles of tumor motes cell proliferation. suppressing UPGs in this deadly disease remain to be eluci- dated. In this study, we conducted a systematic silencing of Cell cycle the E1, E2, E3, and deubiquitinases in NSCLC cells to identify To assess the cell cycle distribution, cells were harvested and tumor suppressing UPGs that were crucial for lung cancer cell washed in phosphate-buffered saline (PBS), fixed in 70% proliferation. ethanol and incubated at 4 °C overnight. Cells were centri- Materials and methods fuged and washed with PBS containing 1% FBS, followed by treatment with 1% RNase A for 15 min at 37 °C and staining Patient samples with 50 μg/mL propidium iodide (Sigma-Aldrich, St. Louis, MO, USA). The fluorescence intensity was measured by flow The study was approved by the local Research Ethics cytometry (FACSVantage Diva, BD Biosciences, San Jose, CA, Committees of all the participating sites; all lung cancer sam- USA). ples were collected with informed consent. The diagnosis of Antibodies lung cancer was confirmed by at least two pathologists. Tissue samples were obtained at the time of surgery and quickly fro- The antibodies used included mouse anti-β-actin (#A5441; zen in liquid nitrogen. Tumor samples contained a tumor cel- Sigma-Aldrich; 1:5000 for Western blot), mouse anti-HA lularity of greater than 60%, and the matched control samples (#AE008; ABclonal, Cambridge, MA, USA; 1:2000 for Western contained no tumor cells. blot), rabbit anti-ubiquitin-like 3 (UBL3) (#A4028; Abclonal; 1:1000 for Western blot and 1:100 for immunohistochemistry Cell culture and siRNA library (IHC)), rabbit anti-Ki67 (#ab15580; Abcam, Cambridge, MA, USA; 1:400 for IHC), rabbit anti-p27 (#sc-528; Santa Cruz The NSCLC cell lines A549, H1975, and H460 were obtained Biotechnology, Santa Cruz, CA, USA; 1:1000 for Western blot), from the American Type Culture Collection (ATCC; Manassas, rabbit anti-Cyclin D1 (#2922; Cell Signaling Technology, Beverly, VA, USA). These cells were cultured in Dulbecco’s Modified MA, USA; 1:1000 for Western blot), and mouse anti-Cyclin E Eagle’s Medium (DMEM) or Roswell Park Memorial Institute (#4129; Cell Signaling Technology; 1:1000 for Western blot). (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL strepto- The siRNA and plasmid transfection mycin (GIBCO BRL, Grand Island, NY, USA). Human siG- ENOME SMARTpool siRNA libraries, containing 696 UPGs The siRNA was purchased from GenePharma Co., (Catalog numbers G-004705-05, G-005615-05, G-005625-05, Ltd. (Shanghai, China), and the sequences were as fol- G-005635-05), were purchased from Thermo Scientific lows: UUCUCCGAACGUGUCACGUTT (siNC); 78 Zhao et al. UBL3 in non-small cell lung cancer

GAGAGUAAUUGUUGUGUAA (#siUBL3-1); and Golden Bridge Biotechnology Co., Ltd., Beijing, China) and CGGCGGAUAUGAUAAAUUU (#siUBL3-2). The HA-UBL3 hematoxylin. vector was constructed based on the pCS2 plasmid. Cells were transfected with siRNA or plasmids using Lipofectamine 3000 Online data availability reagent (Invitrogen, Carlsbad, CA, USA). The Oncomine14 cancer microarray database (www. Lentivirus-mediated cell transfection and oncomine.org), including the Hou Lung, Selamat Lung, transduction Su Lung, StearmanLung, Landi Lung, Okayama Lung, Lee lung, Kuner Lung, and Zhu Lung datasets, with the accession For lentiviral particle production, pCDH-UBL3 constructs codes GSE19188, GSE32867, GSE7670, GSE2514, GSE10072, were co-transfected with psPAX2 and pMD2G into HEK293T GSE31210, GSE8894, GSE10245, and GSE14814, respectively, cells. The culture medium was replaced with fresh medium were used. The transcriptome data and clinical data of 517 after 6 h, and supernatant was harvested 48 h and 72 h patients with LUAD and 501 patients with LUSC were down- post-transfection. A549-luciferase cells were infected with loaded from the Cancer Genomics Hub (https://xenabrowser. viral particles in the presence of 8 μg/mL Polybrene®. One net/datapages/). TCGA database was accessed using the acces- week after infection, cells were sorted by flow cytometry. sion code phs000178. The online survival analysis software containing the microarray data of patients with NSCLC15 Western blot (http://kmplot.com/analysis/index.php?p=service&can- cer=lung) was used. The correlation of UBL3 expression with Cells were lysed in RIPA buffer supplemented with protease OS was evaluated by entering UBL3 into the database and ana- inhibitor cocktail (Sigma-Aldrich). Proteins (20 μg) were sub- lyzed by setting different clinical parameters, and the Kaplan- jected to 10%–15% sodium dodecyl sulfate polyacrylamide gel Meier survival plots and log-rank P values were obtained from electrophoresis (SDS-PAGE), electrophoresed, and transferred the webpage. The baseline demographic characteristics of the to nitrocellulose membranes. After blocking with 5% nonfat patients are shown by Gyorffy et al.15. milk in Tris-buffered saline, membranes were washed and incu- bated with the indicated primary and secondary antibodies. Statistical analysis Immunoreactions were detected using a Luminescence Image Analyzer LAS 4000 (GE, Fairfield, CO, USA). All statistical analyses were conducted using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). All experiments Animal studies were repeated at least three times, and the data were presented as the mean ± SD unless otherwise noted. Differences between Animal studies were approved by the Institutional Review groups of data were evaluated for significance using Student’s Board of the Chinese Academy of Medical Sciences t-test for unpaired data or one-way analysis of variance Cancer Institute and Hospital, with the approval ID of (ANOVA). The survival curve for each group was estimated by NCC2019A188. The methods were performed in accordance the Kaplan-Meier method and log-rank test. P values less than with the approved guidelines. Six-week-old SCID-beige mice 0.05 indicated statistical significance. were maintained under specific pathogen-free (SPF) condi- tions. The mice were numbered, injected into the lateral tail veins with A549-luciferase cells (5 × 105) stably expressing Results pCDH-UBL3 or pCDH-control plasmid, and randomized into groups (n = 13 per group). After 45 days, tumors were Identification of 11 tumor suppressing UPGs monitored with the IVIS Spectrum Imaging System (Caliper in NSCLCs Life Sciences; Hopkinton, MA, USA). For IHC, sections were fixed in formalin and embedded in paraffin, incubated To systematically identify UPGs that are crucial for lung can- with primary antibodies overnight and incubated with the cer cell proliferation, we conducted a systematic silencing of indicated secondary antibodies. Immunoreactions were the UPGs in NSCLC cell lines. The 696 siRNAs for UPGs in detected using 3,3′-diaminobenzidine (DAB, Zhongshan the Dharmacon human siGENOME SMARTpool library were Cancer Biol Med Vol 17, No 1 February 2020 79 transfected into the NSCLC lines A549 and H1975. Cell viabil- paired LUADs and 50 paired LUSCs (Supplementary Table ity was measured 72 h after transfection, and robust Z-scores S1; Figure 1B and 1C), and reported that the expressions from duplicate experiments for each SMARTpool were deter- of five genes (UBL3, TRIM22, UBE2G2, MARCH1, and mined13. While genes with a Z score ≤ -2 have been analyzed RNF10) were downregulated in these NSCLCs. The associ- in other work16, UPGs with a Z-score ≥ 2 (indicating that ation between the expression levels of these five genes and cell proliferation was increased when the gene was silenced) the OS of the patients was analyzed using online survival were assessed here and 11 UPGs were identified (Figure analysis software15 (http://kmplot.com/analysis/index. 1A and Supplementary Figure S1). These included UBL3, php?p=service&cancer=lung). We found that the expression USP26, UBL4, UBE2G2, FBXO42, RNF10, TRIM6, LOC51136, of four genes, UBL3, TRIM22, UBE2G2, and MARCH1, was RNF113A, MARCH1, and TRIM22. positively associated with the OS of the patients, and the median OS of patients with high levels of these genes was Unveiling UBL3 in NSCLCs significantly longer than that of patients with low levels of these genes (Figure 1D–G). In TCGA datasets, the expres- We analyzed the expression levels of the above UPGs in sion levels of the four genes in tumors were lower than those The Cancer Genome Atlas (TCGA) datasets containing 58 in normal tissues at the mRNA level (Figure 1H), and UBL3

AB A549 H1975 58 paired LUADs 50 paired LUSCs TRIM22 UBL3 UBL4A TRIM22 USP26 UBE2G2 RNFT1 MARCH1 TRIM6 RNF10 RNF10 Z- FBXO42 MARCH1 Score RNFT1 4.0 1g (fold change) UBE2G2 TRIM6 3.5 FBXO42 1 3.0 RNF113A RNF113A UBL4A 0 2.5 –1 UBL3 2.0 USP26 CDE 108 paired NSCLCs 1.0 1.0 20 14 0.8 UBL3 P = 3.3e-16 0.8 TRIM22 P = 2.9e-11 8 2 0.6 0.6 2.0 1.5 0.4 0.4 Probabilit y 1.0 Expression Probabilit y Expression Fold change 0.2 0.2 (tumor/normal) 0.5 Low (n = 963) Low (n = 963) 0 0.0 High (n = 963) 0.0 High (n = 963) UBL3 050 100 150 200 050 100 150 200 RNF10 RNFT1USP26TRIM6 UBL4A TRIM22UBE2G2 FBXO42 MARCH1 RNF113A Time (month) Time (month) FG H UBL3 TRIM22 UBE2G2 MARCH1 1.0 1.0 14 14 12 10 UBE2G2 P = 9.7e-10 MARCHI P = 9.8e-06 *** *** *** *** 0.8 0.8 8 12 12 11 0.6 0.6 6 10 10 10 0.4 0.4 4 Probabilit y Expression Probabilit y Expression 0.2 0.2 8 8 9 Low (n = 572) Low (n = 574) 2 High (n = 573) High (n = 571) RNAseqV2 log2(x+1) 0.0 0.0 6 6 8 0 050 100 150 200 050 100 150 200 Paired TumorNormal TumorNormal TumorNormal TumorNormal Time (month)Time (month) n = 108 108 108 108 108 108 108 108

Figure 1 Identification of UBL3 as a potential tumor suppressor in non-small cell lung cancer (NSCLC) cells. (A) Heat map showing 11 candidates with Z-scores ≥ 2 in both A549 and H1975 cells. (B, C) Heat map and scatter plot of the expression of the 11 candidates in 58 paired lung adenocarcinoma (LUAD) tissues and 50 paired lung squamous cell carcinoma (LUSC) tissues. The fold change indicates the tumor/normal ratio. (D–G) The association between the expression levels of the four genes and the prognosis of patients as determined by online survival analysis software. (H) The expression levels of the four genes in TCGA datasets. ***P < 0.0001. 80 Zhao et al. UBL3 in non-small cell lung cancer

ABHou lung Selamat lung CDSu lung Stearman lung EFLandi lung Okayama lung 3 6 4 3 5 7 2 4 5 3 2 6 1 4 1 3 5 0 2 3 0 2 4 –1 1 –2 2 –1 1 3

Normalized UBL3 expression –3 1 0 –2 0 1 Tumor Normal Tumor Normal Tumor Normal Tumor Normal Tumor Normal Tumor Normal n = 72 65 58 58 27 30 20 19 58 49 226 20

G NSCLC HILUAD LUSC JKLUAD LUSC 15 15 15 14 14

12 12

10 10 10 10 10

8 8 RNAseqV2 log2(x+1) RNAseqV2 log2(x+1) RNAseqV2 log2(x+1) RNAseqV2 log2(x+1) RNAseqV2 log2(x+1)

5 5 5 6 6 Tumor Normal Tumor Normal Tumor Normal Paired Tumor Normal Paired Tumor Normal n = 1018 110 517 59 501 51 58 58 50 50

L MO 800 NSCLC (n = 1017) 8 3.0 2.5 700 632 6

l 2.0 600 4 1.5 500 2 1.0 400 2.0 1.0 300 1.5 0.8 200 1.0 0.6 UBL3 protein leve

100 (normalized to actin) 0.4 0.5 Cases with copy number loss 0

Normalized UBL3 expression 0.2 0 0 UBL3 USP26 UBL4A RNF10 TRIM6 RNFT1 Paired Tumor Normal Paired Tumor Normal UBE2G2FBXO42 TRIM22 RNF113A n = 32 32 n = 45 45

NPG11 G13 G15 G17 Q Case 1, UBL3 Case 1, UBL3 14 P = 0.355 T NT NT NT N 12 UBL3 10

Actin Tumor 8 6 G57 G67 G73 105 4 T NT NT NT N 2 UBL3 Immunoreactivity scor e

Normal 0 Actin Paired Tumor Normal n = 14 14

Figure 2 The expression of UBL3 in patients with NSCLC in the Oncomine and TCGA datasets. The expression of UBL3 in the Hou Lung (A), Selamat Lung (B), Su Lung (C), Stearman lung (D), Landi Lung (E), and Okayama lung (F) Oncomine datasets and the NSCLC (G), lung adenocarcinoma (LUAD) (H), and lung squamous cell carcinoma (LUSC) (I) of TCGA datasets is shown. (J, K) UBL3 expression levels in tumor tissues and the counterpart adjacent normal lung tissues from the TCGA LUAD (J) and LUSC (K) datasets. (L) Copy number loss analysis of 10 candidates in the TCGA database. (M–Q) The expression of UBL3 was tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (M), Western blot (N, O), and immunohistochemistry (P, Q) assays of lung cancer patients of our cohort. Size bar, 0.5 mm. The densitometry analysis of the Western blot results (O) and the immunoreactivity score of UBL3 were calculated (Q). P values, Student’s t-test. ***P < 0.0001. Cancer Biol Med Vol 17, No 1 February 2020 81 represented the most significant one (Figure 1H). Therefore, Table 1 Summary of the baseline demographic characteristics of the 86 patients UBL3 was selected for further investigation. Characteristics Cases, n UBL3-low, n (%) P The expression of UBL3 in NSCLC Total 86 59 (68.6)

Gender To evaluate the expression of UBL3 in NSCLC, the Oncomine Cancer Microarray Database14 (www.oncomine.org) was Male 48 36 (75) 0.402 explored. In the Hou Lung17, Selamat Lung18, Su lung19, Female 20 13 (65) 20 21 22 Stearman Lung , Landi Lung , and Okayama Lung data- Not recorded 18 10 (55.6) sets, the UBL3 mRNA levels were significantly reduced in Age (years) tumor tissues compared to those in the counterpart normal < 65 47 33 (70.2) 0.691 lung tissues (Figure 2A–F). We investigated UBL3 RNA levels in TCGA dataset containing 1, 018 NSCLC tumor samples and ≥ 65 20 15 (75) 110 normal lung specimens and found significantly reduced Not recorded 19 11 (57.9) expression of UBL3 in NSCLC tumor samples compared to Smoking status that in normal lung tissue samples (Figure 2G). In both lung Smoker 39 27 (69.2) 0.547 adenocarcinoma (LUAD, n = 517) and lung squamous cell car- cinoma (LUSC, n = 501), the expression of UBL3 was signif- Non-smoker 29 22 (75.9) icantly higher in normal tissues than in tumor lung samples Not recorded 18 10 (55.6)

(Figure 2H and 2I; P < 0.0001). In paired tumor adjacent sam- Histology ples from TCGA datasets, UBL3 expression in tumor tissues LUAD 39 29 (74.4) 0.729 was lower than that in the adjacent lung samples (Figure 2J and 2K, P < 0.0001). In addition, the UBL3 copy number loss LUSC 23 18 (78.3) was found in 632 (62.1%) of 1,017 TCGA patients (Figure 2L). LUAD + LUSC 4 0 (0)

We tested the expression of UBL3 in 86 NSCLCs (Table 1) by SCLC 2 2 (100) quantitative reverse transcription polymerase chain reaction Not recorded 18 10 (55.6) (qRT-PCR) (Figure 2M), Western blot analysis (Figure 2N and 2O), and immunohistochemistry (IHC) (Figure 2P TNM Stage and 2Q), and observed a decrease in UBL3 expression in I-II 36 23 (63.9) 0.150 tumor tissues of 59/86 (68.6%) patients, demonstrating the III-IV 30 24 (80) suppressed expression of UBL3 in NSCLCs. Not recorded 20 12 (60) UBL3 expression and prognosis of smoker LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma. P values were calculated using a two-sided Fisher’s exact test. patients with NSCLC “UBL3-low” indicates that the levels of UBL3 were lower in tumor tissues than in normal tissues. The association between the UBL3 expression levels and tobacco smoking status of the patients was explored in TCGA UBL3 had much better prognosis (Figure 3C and 3D). These and Oncomine databases. In TCGA datasets, the expression of results suggest that the expression of UBL3 in NSCLC may be UBL3 was significantly higher in non-smoking NSCLC patients modulated by tobacco smoke and related carcinogens and thus than in former and current smoking NSCLCs (Figure 3A). involved in lung carcinogenesis. This possibility could be fur- 23 In the Oncomine Lee Lung dataset , UBL3 in non-smoking ther investigated in the future. patients was also higher than in smoking patients with NSCLC (Figure 3B). We analyzed the potential association between The association between UBL3 expression and UBL3 expression and patient prognosis using the online clinical characteristics survival analysis software. The results showed that smoking patients with NSCLC with high levels of UBL3 had a favora- We also explored the association between UBL3 expression ble prognosis, but non-smoking patients with high levels of levels and TNM stage in patients in TCGA database, and the 82 Zhao et al. UBL3 in non-small cell lung cancer

ABTCGA Lee lung CD P = 0.003 3 8 1.0 1.0 P = 0.026 2 0.8 0.8

y NSCLC, smokers y NSCLC, non-smokers 6 P = 0.043 P = 9.1e-06 1 0.6 0.6 0.4 0.4

0 Probabilit Probabilit 4 0.2 Expression 0.2 Expression –1 Low (n = 410) Low (n=102) 0.0 High (n = 410) 0.0 High (n = 103)

Normalized UBL3 expression –2 2 050 100 150 200 050 100 150 Smoke Never Former Current Never Smoker Time (month)Time (month) n = 11 143 31 38 100 EF TCGA, NSCLC 1.0 3 P = 0.01 Stage 1 Stage 2 Stage 3 0.8 2 P = 3.7e-09 P = 0.015 P = 0.35 0.6 1 0.4

0 Probabilit y 0.2 Expression Expression Expression –1 Low (n = 288) Low (n = 122) Low (n = 35) 0.0 High (n = 289) High (n = 122) High (n = 35)

Normalized UBL3 expression –2 050 100 150 050 100 150 050 100 150 Stage IIIIII IV Time (month) Time (month) Time (month) n = 105 38 37 6 GHIJ K TCGA, NSCLC Kuner lung Zhu lung 1.0 1.0 14 10 2 P = 0.06 P = 0.056 LUAD TCGA, LUAD 0.8 P = 3.7e-09 0.8 P = 0.30 12 1 0.6 0.6 8 Low (n = 239) High (n = 240) 10 0 0.4 0.4 Probabilit y 6 0.2 Expression 0.2 8 –1 Low (n = 3 60) 0.0 High (n = 360) RNAseqV2 log2(x+1) 0

Normalized UBL3 expression 050 100 150 200 050 100 150 200 250 6 4 –2 Female Male Female Male Female Male Time (month) Time (month) n = 393 579 14 44 23 67

Figure 3 The association between UBL3 expression and clinical characteristics. The expression of UBL3 in smoking and non-smoking patients with NSCLC in TCGA dataset (A) and Oncomine Lee Lung dataset (B). UBL3 expression and prognosis of smoking (C) and non-smoking (D) patients with NSCLC as determined by online survival analysis software. (E) The expression of UBL3 in patients with NSCLC at different stages. (F) Overall survival (OS) of patients with NSCLCs at different stages with high or low expression of UBL3. (G) Expression of UBL3 in female and male patients with NSCLC in TCGA (G) and the Oncomine datasets Kuner Lung (H) and Zhu Lung (I). The Kaplan-Meier survival curve of LUAD patients with high or low levels of UBL3 expression as determined by online survival analysis software (J) and TCGA database analysis (K). ***P < 0.0001. data showed that the expression of UBL3 was significantly lower expression of UBL3. The expression of UBL3 was sig- higher in stage I NSCLCs than in stage II and III NSCLCs nificantly higher in female NSCLC patients than in male (Figure 3E). The prognostic significance of UBL3 was fur- NSCLC patients in TCGA dataset (Figure 3G). In Oncomine ther analyzed using stage-matched samples. Among stage I datasets24,25, UBL3 expression in female patients was also and stage II patients, those with higher expression of UBL3 higher than in male patients (Figure 3H and 3I), although had much longer survival time than those with lower levels the difference was not statistically significant, and was pos- of UBL3 (Figure 3F). Among patients with stage III NSCLCs sibly attributed to less smoking in women than in men. (Figure 3F, right panel), those with higher UBL3 had slightly Analysis with online survival analysis software indicated that but not statistically significantly shorter OS than those with LUAD patients with lower UBL3 expression had significantly Cancer Biol Med Vol 17, No 1 February 2020 83 worse prognosis than those with higher UBL3 expression Knockdown of UBL3 promotes lung cancer cell (Figure 3J). Similarly, in TCGA datasets, LUAD patients with proliferation lower UBL3 expression had poorer prognosis, although the difference was not statistically significant (Figure 3K). These We found that silencing UBL3 accelerated cell proliferation, data demonstrated that UBL3 might play a role in the patho- with Z-scores of 4.22 and 3.42 in A549 and H1975 cells, respec- genesis of NSCLC. tively (Figure 1A and Supplementary Figure S1). We verified

ABC A549 H1975 A549 H460 A549 H1975 siUBL3 NC 1# 2# NC 1# 2# 25 5 HA-UBL3 –++ –

UBL3 ) HA 5 20 4 SiNC Actin 10 SiUBL3-1# Actin ( × 15 3 8 6 P = 0.02 P = 0.04 8 10 10 2 8

) 6 ) 5 6 4 5 6 10

Viable cells 5 1 10 4

( × 4 ( × 4 2 0 0 2 2 0 24 48 72 0244872 2 Relative cell numbe r

Time post transfection (h) Relative cell numbe r 0 0 0 0

D A549 H460 E 15 20 Foci formation Soft-ager A549 )

5 HA-vector 15 HA-UBL3 siUBL3 NC 1# 2#

( × 10 10 UBL3

P = 0.05 P = 0.02 NC

10 si 5 Actin 5 Foci formation Viable cells 400 P = 0.038 P = 0.044 0 0 0 24 48 72 0244872 300 Time post transfection (h) 200 A549: siNC siUBL3-1# siUBL3-2# F UBL3 - 1# 100 si G1: 75.44% G1: 63.19% G1: 67.6% No. of colonies 100 G2/M: 11.45% G2/M: 6.21% G2/M: 10.28% 0 S: 13.11% S: 30.6% S: 22.02% Soft-agar 80 P = 0.35 300 P = 0.05 60 G1

Number G2 200 40 S UBL3 - 2#

20 si 100

0 No. of colonies 0 40 80 120 030 60 80 120 04080 120 0 siUBL3 NC 1# 2# H1975 A549 H1975 G1: 67.94% G1: 60.96% G1: 62.82% G 100 G2/M: 17.39% G2/M: 11.88% G2/M: 11.87% siUBL3 NC 1# 2# NC 1# 2# S: 14.67% S: 27.17% S: 25.31% 80 UBL3 60 Cyclin E Number 40 Cyclin D1 20 p27 0 0 40 80 0 100 200 050 200 Actin Channels (FL2-A)

Figure 4 UBL3 inhibits NSCLC cell proliferation. (A) A549 and H1975 cells were transfected with siUBL3 (1# and 2#) and lysed 72-h later for the detection of UBL3 expression by Western blot assays (upper panel) and assessment of cell viability (lower panel). Error bars, SD. (B) A549 and H1975 cells were transfected with siUBL3-1, and cell proliferation was assessed by a Trypan Blue dye exclusion assays. (C, D) A549 and H460 cells were transfected with HA-tagged UBL3, and cell proliferation was assessed by a Trypan Blue exclusion assay. (E) Foci formation and soft agar assays of A549 cells transfected with siUBL3. Error bars, SD; P values, Student’s t-test. (F) A549 and H1975 cells were transfected with siUBL3 and the cell cycle distribution was assessed by propidium iodide staining of DNA followed by flow cytometric analysis. (G) A549 and H1975 cells were transfected with siUBL3, lysed 72 h later, and the lysates were subjected to Western blot assay using indicated antibodies. 84 Zhao et al. UBL3 in non-small cell lung cancer the effects of siRNA against UBL3 (siUBL3) on lung cancer D1, and upregulation of Cyclin E (Figure 4G), indicating that cells, and found that siUBL3 treatment resulted in a reduc- UBL3 played a role in the control of the replication checkpoint. tion of UBL3 protein expression and increased viability of A549 and H1975 cells (Figure 4A). Knockdown of UBL3 led UBL3 inhibits tumor growth in vivo to a significant increase in cell growth (Figure 4B). In contrast, exogenous expression of UBL3 (by transient transfection) sig- To investigate the role of UBL3 in tumor growth in vivo, the nificantly inhibited the proliferation (Figure 4C) and growth pCDH-UBL3 plasmid was transfected into A549-luciferase (Figure 4D) of cells. We further showed that knockdown of cells (Figure 5A), which were then injected into SCID-beige UBL3 promoted the colony-forming activity of A549 cells mice via the tail vein (n = 13 for each group). To characterize (Figure 4E). Silencing UBL3 in H1975 cells led to cell cycle the growth of the xenograft tumors, the luciferase intensity in arrest at the S phase (Figure 4F), downregulation of p27, Cyclin the mice was measured by an IVIS Spectrum system 45 days

A B A549-luc pCDH-vector pCDH-UBL3 ph/s 15 4.0 2.5 ) y 10 2 2.0 3.0 5 1.5 2.0 2 ph/s/mm 1.0 1.0 4 expression ∗∗∗ 1 10 0.5 Average intensit Normalized U BL 3 ( × 4 0 0 ×10 pCDH-UBL3 –+ pCDH-UBL3 –+ E HE UBL3 Ki67 CD

) 100 pCDH-vector pCDH-UBL3 80 no. 1# 2# 3# 1# 2# 3#

n = 13 pCDH - vector 60 UBL3 40 pCDH-vector 20 pCDH-UBL3 *** Actin

Percent survival (% 0 SCID beige mice 0 30 60 90 120 150 pCDH - UBL3 Time (day) F A549-luc: pCDH-vector pCDH-UBL3 G A549-luc G1 G1: 60.79% G1: 70.08% 100 G2/M: 21.03% G2/M: 21.8% G2/M S: 18.18% S: 8.12% 80 80 S )

60 60

40 Number 40 Percentage (%

20 20

0 0 pCDH-UBL3 –+ 020406080 120 02040 60 80 120 Channels (FL2-A)

Figure 5 UBL3 suppresses lung cancer in vivo. (A) qRT-PCR analysis of UBL3 mRNA levels in pCDH-UBL3-expressing A549-luciferase cells. (B) A total of 5 × 105 pCDH-UBL3-expressing A549-luciferase cells were injected into SCID-beige mice and the relative luciferase intensity was determined by an IVIS Spectrum system at the indicated time point (B). Error bars, SD; P values, Student’s t-test. The Kaplan-Meier survival curve of the mice is shown (C). P value, log-rank test. Mice were sacrificed, and lung tissues were lysed for Western blot assays using the indicated antibodies (D) or subjected to hematoxylin-eosin (HE) or immunohistochemical staining (E). Scale bar, 1 mm for HE and 0.5 mm for IHC. (F, G) A549-luc cells with pCDH-vector or pCDH-UBL3 were assessed by propidium iodide staining of DNA followed by flow cytometric analysis (F), and cell cycle distribution was assessed (G). Cancer Biol Med Vol 17, No 1 February 2020 85 after cell inoculation. We found that UBL3 significantly Here, we found that UBL3 expression was reduced in tumor decreased the tumor burden (Figure 5B) and extended the life samples from both TCGA and Oncomine datasets and in 86 span of the mice (Figure 5C). Western blot assays of tumor NSCLC samples from our laboratory (Figure 2, Table 1). lysates indicated overexpression of UBL3 in vivo (Figure 5D). UBL3 expression levels were much lower in patients with Hematoxylin-eosin (HE) and Ki67 staining of lung sections stage II or III NSCLC than in patients with stage I NSCLC confirmed the tumor-inhibitory effect of UBL3 (Figure 5E). (Figure 3E). LUAD patients with higher levels of UBL3 had Cell cycle analysis showed that overexpression of UBL3 more favorable prognosis than those patients with lower lev- induced arrest at G1 phase, without accumulation of sub-G1 els of UBL3 (Figure 3). We investigated the function of UBL3 content (Figure 5F and 5G). in NSCLC cells and demonstrated that silencing of UBL3 pro- moted, whereas overexpression of UBL3 suppressed, NSCLC Discussion cell proliferation in vitro and in vivo (Figure 4 and Figure 5). These data indicated that UBL3 could be a tumor suppressor In this study, we carried out an siRNA screen for 696 UPGs in NSCLCs. However, why UBL3 was suppressed in tumor found in the , and reported 11 candidates that samples and how it suppressed cell proliferation, warrant fur- were able to promote cell proliferation when they were silenced ther investigation. in A549 and H1975 cells (Figure 1). In TCGA database, the Tobacco is responsible for about 85% of lung cancer deaths expressions of four potential tumor suppressor genes (UBL3, (1.53 million) each year worldwide1. Tobacco smoke causes TRIM22, UBE2G2, and MARCH1) were significantly down- genomic mutations in cancers36; promotes cell proliferation; regulated in tumor samples compared to that in normal lung inhibits programmed cell death; facilitates angiogenesis, inva- tissues. Using online survival analysis software15, we found that sion and metastasis; and enhances tumor-promoting inflam- the expression levels of these four genes were positively asso- mation37,38. We found that the expression of UBL3 was sig- ciated with the OS of patients with NSCLC, with UBL3 as the nificantly higher in non-smoking than in smoking patients most significant one. These results provided rationales for us and that non-smoking patients with NSCLC with high levels to further investigate the role of UBL3 in lung carcinogenesis. of UBL3 had much more favorable prognosis than those who UBL3 has been identified inDrosophila melanogaster26 and smoked (Figure 3). The effects of tobacco smoke on UBL3 is a membrane protein localized by prenylation27. UBL3 acts expression need to be investigated in the future. as a post-translational modification factor that regulates pro- Collectively, the above results suggested that UBL3 may be tein sorting to small extracellular vesicles (sEVs)28. In melano- involved in lung carcinogenesis by altering the expression of cytic cells, UBL3 may be regulated by MITF29. A genome-wide oncoproteins/tumor suppressors, thus affecting critical path- association study demonstrated that UBL3 harbors a sus- ways to promote cell proliferation and disease progression. ceptibility locus for biliary atresia30. Additionally, UBL3 was These possibilities and whether UBL3 could be a therapeutic identified to harbor a novel susceptibility locus for BRCA1/2- target for lung cancer warrant further investigation. negative, high risk breast cancers in Asian women31. UBL3 was upregulated in human prostate cancer cells (LNCaP Acknowledgments cells) exposed to silvestrol32. UBL3 was identified as part of a 7-gene signature (UBL3, FGF3, BMI1, PDGFRA, PTPRF, This work was supported by the National Key Research and RFC4, and NOL7) for predicting relapse and survival in ear- Development Program of China (Grant No. 2016YFC0905501), ly-stage patients with cervical carcinoma33. Promoter hyper- the National Natural Science Funds for Distinguished Young methylation and downregulation were found in UBL3 in a Scholar (Grant No. 81425025), the Key Project of the National northeast Indian population with esophageal cancer34. An Natural Science Foundation of China (Grant No. 81830093), in-frame fusion of MAP3K8-UBL3 was reported as a distinct the CAMS Innovation Fund for Medical Sciences (Grant No. genetic driver in a unique subgroup of spitzoid neoplasms35. CIFMS; 2019-I2M-1-003), and the National Natural Science Previous studies showed that UBL3 was abnormally expressed Foundation of China (Grant No. 81672765 and 81802796). in BRCA1/2-negative, high risk breast cancers (BRCAX)31, The study sponsor had no role in the design of the study; the cervical carcinoma33, and esophageal cancer34. However, the data collection, analysis, or interpretation; the writing of the role of UBL3 in lung cancer remains poorly understood. article; or the decision to submit for publication. 86 Zhao et al. UBL3 in non-small cell lung cancer

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Supplementary materials

Table S1 Summary of the baseline demographic characteristics of 4.5 the 108 patients with non-small cell lung cancer (NSCLC) in TCGA A549 4 database H1975 3.5 Characteristics Cases, n UBL3-low, n (%) P 3 Total number 108 76 (70.4) 2.5 Age (years) 2 Z scor e < 65 41 26 (63.4) 0.216 1.5 1 ≥ 65 67 50 (74.6) 0.5 Gender 0

Male 51 36 (70.6) 0.009 UBL3 UBL4 USP26 RNF10 TRIM6 UBE2G2 FBXO42 TRIM22 RNF113AMARCH1 Female 57 26 (45.6) LOC51136

Smoking status Figure S1 The 11 UPGs that have a Z score of ≥ 2. Smoker 95 72 (75.8) 0.275

Non-smoker 7 4 (57.1)

Not recorded 6 4 (66.7)

Histology

LUAD 58 30 (51.7) 4.86E-06

LUSC 50 46 (92)

TNM stage

I-II 85 57 (67.1) 0.092

III-IV 21 18 (85.7)

Not recorded 2 1 (50)

Anatomic subdivision of lung neoplasm

Upper 63 43 (68.3) 0.619

Lower 37 27 (73)

Middle 4 4 (100)

Not recorded 4 2 (100)

Vital status

Living 56 32 (57.1) 0.002

Dead 52 44 (84.6)

P values were calculated using a two-sided Fisher’s exact test. “UBL3-low” indicates that the levels of UBL3 were lower in tumor tissues than in normal tissues. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma.