TEX19 Promotes Ovarian Carcinoma Progression and Is a Potential Target for Epitope Vaccine Immunotherapy T

Life Sciences 241 (2020) 117171

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TEX19 promotes ovarian carcinoma progression and is a potential target for epitope vaccine immunotherapy T

Zhaoxu Xua,b,c,d, Haichao Tanga,b,c,d, Tianshu Zhange,f, Mingli Suna,b,c,d, Qiang Hana,b,c,d, ⁎ ⁎ Jiao Xua,b,c,d, Minjie Weia,b,c,d, , Zhaojin Yua,b,c,d, a Department of Pharmacology, School of Pharmacy, China Medical University, No.77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning 110122, PR China b Liaoning Key Laboratory of molecular targeted anti-tumor drug development and evaluation, China Medical University, No.77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning 110122, PR China c Liaoning Cancer immune peptide drug Engineering Technology Research Center, China Medical University, No.77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning 110122, PR China d Key Laboratory of Precision Diagnosis and Treatment of Gastrointestinal Tumors, Ministry of Education, China Medical University, No.77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning 110122, PR China e Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, No.1 Tian Tan Xi Li, Dongcheng District, Beijing 100050, PR China f No.9, Dongdan Santiao, Dongcheng District, Beijing 100730, PR China

ARTICLE INFO ABSTRACT

Keywords: Aims: Testis Expressed 19 (TEX19) is one of cancer/testis antigens identified in recent years and is related to the TEX19 oncogenesis and progress of several cancers. This study aimed to reveal the role of TEX19 in ovarian cancer (OC) Ovarian cancer and searched for potential candidate epitope peptides of TEX19 to facilitate clinical application. Proliferation Main methods: TEX19 levels were evaluated by immunohistochemistry (IHC) in 98 human ovarian tissue sam- Invasion ples. The correlation of TEX19 levels with patients' clinicopathological features was assessed. Quantitative real- Metastasis time polymerase chain reaction (qRT-PCR) and western blotting analysis were utilized to detect TEX19 levels in Epitope peptide ovarian cell lines and TEX19-deficient cells. The level of TEX19 in OVCAR-3 and A2780 was knocked down by small interfering RNA (siRNA), and loss-of-function assays were used to determine the biological effects of TEX19 on the proliferation, migration, and invasion of OC cells. Subsequently, candidate epitope peptides from TEX19 were predicted and verified by the IEDB database, pepsite2 website, MOE software, and T2 cell binding assay. Key findings: TEX19 was significantly upregulated in OC which correlated to higher TNM stage, lymph node involvement, and invasiveness. Knockdown of TEX19 inhibited proliferation, migration, and invasion of OC cells. Additionally, we screened four peptides derived from TEX19 and found TL to be the dominant peptide with the greatest affinity with HLA-A*0201. Significance: Our data indicated a cancer-promoting effect of TEX19 in OC and demonstrated that TL could be a potential candidate for an anti-tumor epitope vaccine of OC, suggesting that TEX19 is a promising biomarker and immunotherapeutic target for OC.

1. Introduction patients in medically rich countries such as the United States and Ca- nada is only 47% [4–7]. In recent years, despite some valuable ad- Ovarian cancer (OC) is one of the most common and lethal malig- vances in diagnosis strategies, surgical techniques, and individual nancies, having the third-highest prevalence, the poorest prognosis, and chemotherapy programs, the early diagnosis and prognosis of patients the highest mortality rate in gynecological malignant tumors [1,2]. with OC are still not satisfactory [8–10]. Therefore, it is extremely ur- Compared with many malignant tumors, OC is more concealed due to gent to ﬁnd functional and predictive biomarkers for designing reliable late-presenting symptoms and a lack of eﬀective screening strategies diagnostic strategies and precise treatment plan. [3]. Even after diagnosis, the 5-year survival rate of ovarian cancer With the studies in the past two decades, cancer/testis antigens

⁎ Corresponding authors at: Department of Pharmacology, School of Pharmacy, China Medical University, No.77, Puhe Road, Shenyang North New Area, Shenyang, Liaoning 110122, PR China. E-mail addresses: [email protected] (M. Wei), [email protected] (Z. Yu). https://doi.org/10.1016/j.lfs.2019.117171 Received 3 September 2019; Received in revised form 3 December 2019; Accepted 11 December 2019 Available online 13 December 2019 0024-3205/ © 2019 Elsevier Inc. All rights reserved. Z. Xu, et al. Life Sciences 241 (2020) 117171

(CTAs) encoded by some germline genes are only found to be present in 2.4. Evaluation of immunohistochemistry adult testis, fetal ovary, and placenta [11]. Many researchers have reported that CTAs showed highly elevated levels in cancer and had a All stained microarray specimens were evaluated in a blinded significant correlation with malignant tumor growth, metastasis, and manner by two experienced researchers (ML S. and ZJ Y.). TEX19 is poor prognosis [12–15]. And many CTAs are immunogenic and nor- mainly localized in the nucleus and cytoplasm of cancer cells. mally exist in immune-privileged sites, so they are considered to be According to the percentage of positively stained tumor cells (0 to suitable targets for therapeutic cancer vaccines and potential bio- 100%) and the score of immunohistochemical staining intensity (no markers for early diagnosis [16–20]. Some CTAs have been studied staining, 0; weak staining, 1; medium staining, 2; and deep staining, 3), intensively as therapeutic vaccines against various cancers [21–24]. we obtain an immune response score (IRS) ranging from 0 to 3. Testis Expressed 19 (TEX19) is a defined CTA encoded by a mammalian specific gene located at 17q25.3 of human chromosome 17 [25–28]. In rodents it is known that the TEX19 paralogues (Tex19.1/ 2.5. Cell lines and culture Tex19.2) function to regulate germline and placental genome stability and RNA expression [29–32], but the function of TEX19 in human Human ovarian surface epithelial cells (HOSEpiC), OVCAR-3, HO- tissues and cancer cells remains poorly defined [25,26]. It's worth 8910, SKOV-3, A2780, and T2 cells were obtained from Bena Bio noting that some studies have reported human TEX19 is related to (Beijing, China). HOSEpiC and OVCAR-3 were cultured in RPMI-1640 cancer, including OC [28,33,34]. However, there is not enough re- medium containing 10% FBS and 1% penicillin/streptomycin. HO- search about clinical significances and function of TEX19 in OC. In this 8910, SKOV-3, and T2 were respectively cultured in DMEM (High study, our data first confirmed the up-regulation of TEX19 protein le- Glucose) medium, McCoy's 5a medium, and IMEM medium, all con- vels in OC and revealed the progression-promoting effect of TEX19 in taining 10% FBS and 1% penicillin/streptomycin. The above cell lines

OC. Furthermore, we predicted and screened one candidate epitope were cultured at 37 °C with 5% CO2. All cell experiments were per- peptide with the promising prospect for immune activation. formed during the logarithmic growth phase of the cells. According to an earlier study, HO-8910 may be contaminated by other cell lines, so 2. Materials and methods we did not choose it in our further functional experiments [35].

2.1. Bioinformatics analysis 2.6. Immunofluorescence The GSEA 3.0 tool analyzed data from the cancer genome atlas (TCGA) to obtain enrichment analysis results to predict and evaluate The HOSEpiC, A2780, OVCAR-3, HO-8910, and SKOV-3 cells were 5 the correlation between TEX19 mRNA expression and biological func- seeded at a density of 1 × 10 in 6-well plates with coverslips, and then fi tion of cell. cultured for 6 h. These cells were xed with 4% paraformaldehyde for 15 min and then permeabilized by 0.1% Triton X-100 (T8200; Solarbio, China) at room temperature for 5 min. After blocked by 5% bovine 2.2. Human tissue specimens serum albumin (BSA) at room temperature for 30 min, these cells were incubated with the primary antibodies against TEX19 (1200, ab185507; In this study, ovarian carcinoma tissue microarray with adjacent Abcam, UK) at 4 °C overnight. Next, these cells were incubated with normal tissue was obtained from US Biomax (chip number: BC11115b), fluorescein isothiocyanate (FITC)-conjugated secondary antibody containing 5 cases of clear cell carcinoma, 62 cases of serous adeno- (E031220, Earthox, USA) in the dark at 37 °C for 1 h, and then were carcinoma with one of which are not available, 12 cases of mucinous sealed by DAPI dissolved in glycerol in the dark for 5 min. The images adenocarcinoma with one of which are not available, 1 case of en- were observed under a laser scanning confocal microscope (×400 dometrioid adenocarcinoma, 10 cases of lymph node metastatic ade- magnification, C2 plus, Nikon, Japan). nocarcinoma, and 10 cases of adjacent normal tissue. In 98 ovarian carcinoma specimens, the average age of the patients was 49.7 ± 10.5 years (range from 22 to 83 years); the original case data of 2.7. Quantitative real-time polymerase chain reaction (qRT-PCR) all patients recorded the patients' age, gender, histotype, TNM stage, and histopathological grade. Total RNA was isolated using TRIzol (R1100; Solarbio, Beijing, China) according to the manufacturer's instruction. RNA was reverse 2.3. Immunohistochemistry (IHC) transcribed to cDNA using PrimeScript™ RT Master Mix Kit (RR036A; TAKARA, Tokyo, Japan) according to the manufacturer's protocol. qRT- Paraffin-embedded tissue microarray (1 mm) was stained using a PCR was performed using TB Green® Premix Ex Taq™ II Kit (RR820A; polymer horseradish peroxidase (HRP) detection system. The micro- TAKARA, Tokyo, Japan). β-actin was amplified as an internal control. array was immersed in xylene for deparaffinization for 30 min and The primers (Sangon, Shanghai, China) are listed in Table 1. Relative −ΔΔ rehydrated in gradient ethanol and ultrapure water, followed by an- expression levels between samples were calculated using the 2 CT tigen retrieval utilizing 0.01 M citrate buffer (pH 6.0) and processing method. the microarray at 80 kPa high pressure for 8 min. The endogenous peroxidases were eliminated with 0.3% H O for 10 min. Subsequently, 2 2 Table 1 the microarray was blocked with 10% goat serum (Solarbio, Beijing, The sequence of gene primer and small interfering RNAs. China) for 30 min at 25 °C. The microarrays were incubated with rabbit anti-TEX19 polyclonal antibody (1:500, ab185507; Abcam, UK) over- Name Primer/interfering sequence night at 4 °C in the constant low-temperature chamber. It was then TEX19 F: 5′-CATGTCCTACCTCTACGCCTCCTG-3′ incubated with HRP-conjugated secondary antibody (1:50, ZDR-5306; R: 5′-TCCCAGTTGTCTTCTTCCCAGTCC-3′ Zhongshan Gold Bridge Biotechnology, Beijing, China) for 30 min at β-actin F: 5′-TCCTCCCTGGAGAAGAGCTA-3′ 25 °C and stained with 3, 3-diaminobenzidine (DAB) for 25 s. The mi- R: 5′-TCCTGCTTGCTGATCCACAT-3′ ′ ′ croarray was counterstained with hematoxylin, dehydrated with re- siRNA1 5 -CTTCAAGGCTGCCTTTCTA-3 siRNA2 5′-CATGGAGATCAGCTAAGCA-3′ verse gradient ethanol. Finally, we use a neutral resin to seal the slide siRNA3 5′-CAGCATGCGGTATGAGGAA-3′ without bubbles.

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2.8. Western blotting the upper chambers containing serum-free medium, and complete medium was added into the lower chambers. Until cells were cultured Each total protein sample from the OC cells and epithelial cells were 48 h, the lower surface of chambers was stained with 0.1% crystal prepared by total protein extraction kit (BB-3101; Bestbio, China). Total violet (C8470, Solarbio, China). The cells on the lower surface of protein levels were measured by BCA assay kit (P0010S; Beyotime chambers were counted under a microscope in five random fields Biotechnology, China). Next, the equal quantity of protein from each (magnification, ×400). sample was separated by 12% SDS-PAGE and transferred to 0.22μm polyvinylidene fluoride membranes. After blocked with TBST solution 2.13. T cell antigen epitope prediction (0.1% Tween 20) with 5% (w/v) non-fat milk for 1 h, the membranes were incubated at 4°C overnight with the primary antibodies against For predicting Cytotoxic T cell (CTL) epitopes interacting with HLA- the TEX19 (1:1000, ab90953; Abcam, UK), or β-actin (1:1000, 8457, A*0201, the sequences of human TEX19 was obtained from GenBank, CST, USA). Then, the blots were incubated with HRP-conjugated sec- and the Immune Epitope Database (IEDB, https://www.iedb.org) was ondary antibody (110,000, E030120, EarthOx, USA) for 2 h at room used [36,37]. According to the recommendation of IEDB, the consensus temperature. Electro-chemiluminescence detection system (Pierce, method, consisting of artificial neural network (ANN) [38], stabilized Thermo Fisher Scientific, USA) were used to visualize the bands. The matrix method (SMM) [39], and Scoring Matrices derived from Com- displayed protein blots were detected and analyzed by using ImageJ binatorial Peptide Libraries (CombLib) [40], is selected as MHC class I 1.52a software. binding prediction method. All selected epitope peptides are limited to 9 amino acids in length [41,42]. As guideline, peptides with the half- 2.9. Cell transfection maximal inhibitory concentration (IC50) values < 50 nM are considered high affinity, < 500 nM intermediate affinity and < 5000 nM OVCAR-3 and A2780 cells were seeded at a density of 2 × 105 cells low affinity. In addition to the IC50 values for each peptide, a percentile per well in 6-well plates and cultured for 12 h in medium containing rank is generated by comparing the peptides' IC50 against those of a set 10% FBS. For transient transfection, cells were starved in serum-free of random peptides from SWISSPROT database. A small numbered medium for 2 h and incubated with siRNA (stB0017918A-1-5, percentile rank indicates high affinity. Among a large number of results stB0017918B-1-5, and stB0017918A-1-5; Ribobio, Guangzhou, China), obtained by screening, epitope peptides with IC50 value < 500 nM including 50 nM specific siRNAs against TEX19 or a scrambled negative were considered as candidate epitope peptides [43]. control, and Lipofectamine™ 2000 (11,668,019; Thermo Fisher The Pepsite2 platform (https: //pepsite2.russelllab.orgn) was used Scientific, USA) for 48 h, and were ready for subsequent experiments. to analyze and evaluate the potential of the candidate epitope peptides The siRNA sequences of TEX19 are listed in Table 1. The number of to effectively bind to HLA-A*0201 (PDB code: 3v5k). siRNA treatments per wells depended on the different assays. For ex- Docking of these candidate peptides with HLA-A*0201 (PDB code: ample, siRNA was incubated with cells at least once every 3 days for 3v5k) that went through quick molecular preparation was run with the plate clone formation assay over extended periods. algorithm of the Molecular Operating Environment (MOE2018.0101). Parameters were set as Placement, Triangle matcher; Refinement, Rigid 2.10. CCK-8 proliferation assay Receptor; scoring 1, London dG, poses, 100; and scoring 2, GBVIWSA dG, poses, 10. The idea with the highest S-score was chosen to assess OVCAR-3 and A2780 Cells, which were transfected, were seeded at the interaction of epitope peptides with HLA-A*0201. a density of 2 × 103 cells per well in 96-well plates. Each well contained three repetitions, and PBS was added into unused wells. After 2.14. T2 cell binding assay cultured for 4, 24, 48, 72, or 96 h, cells were incubated with CCK-8 reagent (Biosharp, Hefei, China) at 37 °C for 2 h. Cell proliferation To further confirm the accuracy of bioinformatic analysis, the curves were plotted by measuring optical density (OD) of each plate at ability of the candidate epitope-peptides to bind HLA-A*0201 mole- 450 nm. cules was examined using T2 cells (ATCC CRL-1992TM). 3 × 105 T2 cells were incubated with 100 μM of the synthesized peptides 2.11. Plate clone formation assay (Yuanpeptide, Nanjing, China), the purity of them higher than 95%, and 100 ng/ml of human β2m (M4890, Sigma, USA) in serum-free OVCAR-3 and A2780 cells, which were transfected, were seeded RPMI 1640 medium at 37 °C for 16 h. T2 cells only incubated with into 6 cm petri dishes at a density of 1000 cells, and then cultured for human β2m served as a negative control. Then, the cells were washed 2 weeks until visible clones appeared in petri dishes. Next, these were and the expression of HLA-A*0201 on T2 cells was then tested by fixed with 4% paraformaldehyde for 30 min and dyed with 0.01% bonding with BB7.2 monoclonal antibody (343,304, BioLegend, USA). crystal violet (C8470, Solarbio, China) for 30 min. The clone formation The fluorescence index (FI) was defined as follows: FI = (mean fluor- rate was calculated by counting the number of clones. escence intensity (MFI) with the given peptide − MFI without peptide)/MFI without peptide. FI value of the peptide is higher than 1.5, 2.12. Cell migration and invasion assay which can be considered that the peptide can have a good affinity with HLA-A*0201. To detect cell migration, we used wound-healing assay. In 6-well plates, OVCAR-3 and A2780 cells, which were transfected, were seeded 2.15. Statistical analysis at a density of 2 × 105 cells per well and cultured for 24 h in complete medium. Since a wound was scraped with a pipette tip, the left cells All results were repeated three-time and presented as the mean ± which were washed by PBS were cultured in serum-free medium. After standard deviation (SD). Pearson Chi-square test or Fisher's Exact Test cultured for 0 and 24 h, cells were observed and photographed with were utilized to analyze the relationship between level of TEX19 and Nikon Eclipse TS100 Microscope (Nikon, Japan). other clinicopathological variables. The cut-off value used to define the To investigate invasion and migration activity of OVCAR-3 and low and high level of TEX19 was obtained by the receiver operating A2780 cells, we used the transwell cell invasion assay as followed. The characteristic (ROC) curve analysis. The differences between groups upper surface of each 24-well transwell chamber (3422, Corning, USA) were compared with the independent-samples t-test. P < 0.05 was was coated with or without Matrigel (356,234, Corning, USA). OVCAR- defined as statistically significant. All statistical calculations were 3 and A2780 cells were seeded at a density of 4 × 105 cells per well in conducted by using the SPSS software (SPSS 20.0 Inc., Chicago, IL,

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USA) and GraphPad Prism 7.0 (GraphPad Software, Inc., La Jolla, CA, analysis demonstrated that TEX19 levels in OC tissues were higher USA) was manipulated to draw graphs. compared with adjacent normal tissues (median IRS: 0.9882[0.00–2.55] vs 0.3250[0.00–1.35], P = 0.0061). The range of staining intensity, including no staining, weak staining, medium 3. Results staining, and deep staining, was presented in Fig. S1. In addition, TEX19 levels were investigated in OC cell lines (OVCAR-3, A2780, HO- 3.1. TEX19 is up-regulated in OC tissues and cell lines 8910, and SKOV-3) and one human ovarian epithelial cell line (HOSEpiC). As presented in Fig. 1C and D, immunofluorescence assay, ff In order to determine TEX19 levels di erences between OC tissues western blotting, and qRT-PCR showed the upregulated protein and fi and normal tissues, we rst detected TEX19 levels in 88 OC tissue mRNA levels of TEX19 in 3 OC cell lines compared with the ovarian specimens and 10 paracancerous tissue specimens by im- epithelial cell line (P < 0.05). Thus, our data suggested that TEX19 munohistochemistry. As shown in Fig. 1 A and B, immunohistochemical

Fig. 1. TEX19 levels are high in OC. Note: (A) Representative micrographs presenting immunohistochemical staining of TEX19 in tumor-adjacent tissues and OC tissues. Magnification: ×100. The boxes marked the magnified regions in the insert (×400). (B) IRS of TEX19 levels in cancer tissues was significantly higher than tumor-adjacent tissues. Redline means the median value. (C) Immunofluorescence analysis of TEX19 protein levels in OC cell lines and human ovarian epithelial cell line. (D) Western blotting analysis of TEX19 protein levels in OC cell lines and human ovarian epithelial cell line. (E) qRT-PCR analysis of TEX19 mRNA levels in OC cell lines and human ovarian epithelial cell line. *P < 0.05, **P < 0.01, and ***P < 0.001. Abbreviations: TEX19, testis expressed 19; OC, ovarian cancer; IRS, immunoreactive score.

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Fig. 2. ROC curves were utilized to determine the cut-off score for the positive level of TEX19 protein. Note: The sensitivity and specificity for each outcome were plotted: (A) Benign and malignant, (B) Age, (C) TNM stage, (D) Depth of invasion, (E) Lymph node involvement, and (F) Grade. Abbreviations: ROC, receiver operating characteristic; TEX19, testis expressed 19; AUC, area under the curve; IHC, immunohistochemistry. levels were elevated in OC tissues and cell lines. Table 2 Association of TEX19 levels with clinicopathological features of serous ovarian adenocarcinoma patients. ff 3.2. Selection of a cut-o value for TEX19 levels Features Categories TEX19 P-valuea

Due to differences in histology, genomics, transcriptomics, and cell Low(%) High(%) origin among different pathologic types of OC, follow-up studies fo- Age(years) ≤50 8(40.6) 16(59.) 0.310 cused on serous ovarian adenocarcinoma. In order to analyze the cor- > 50 8(21.6) 29(78.4) relation between TEX19 levels and clinicopathological factors, the IRS TNM stage I 13(40.6) 9(59.4) 0.008 cut-off value of the high and low protein levels was defined by the ROC II + III + IV 3(10.3) 26(89.7) curves. The sensitivity and specificity for benign and malignant, age at Distant metastasis No 16(27.6) 42(72.4) 0.290 Yes 0(0) 3(100) diagnosis, TNM stage, depth of invasion, lymph node involvement, and Depth of invasion T1 13(40.6) 19(59.4) 0.008 histopathological grade were plotted in Fig. 2. According to area under T2 + T3 3(10.3) 26(89.7) the curve (AUC), the ROC curve analysis with respect to benign and Lymph node involvement No 15(32.6) 31(67.4) 0.014 malignant was used to determine the cut-point of the IRS (cut-point YES 0(0) 14(100) value = 0.475, AUC = 0.798, P = 0.003; Fig. 2A). Based on this cut-off Grade 1 + 2 9(36.0) 16(64.0) 0.125 3 6(25.9) 27(74.1) value, 45 (73.8%) spots of OC tissue showed high TEX19 staining, while only 2 (20%) spots of paracancerous tissue showed high TEX19 Notes: staining. a P-value was obtained by Pearson chi-squared test, likelihood-ratio test, or Fisher's exact test. Bold values indicate statistical significance. Abbreviations: TEX19, testis expressed 19; OC, ovarian cancer. 3.3. Association between TEX19 levels and clinicopathological parameters of patients with serous ovarian adenocarcinoma 3.4. Down-regulation of TEX19 decreased proliferation of OC cells

Subsequently, we further investigated whether there was a corre- To analyze the role of TEX19 in OC, GESA analysis based on clinical lation between TEX19 levels and the clinicopathological features of data from TCGA dataset was performed. As presented in Fig. S2, those patients with serous ovarian adenocarcinoma. Chi-square analysis re- samples with high TEX19 mRNA expression were relevant to enrich- vealed that the rate of samples with high level of TEX19 was increased ment in DNA replication and cell cycle of OC, which indicated that in OC samples with TNM stage (II + III + V; P = 0.005), positive TEX19 might be critical for enhancing the proliferation of OC cells. To lymph node involvement (P = 0.012), and deeper invasion (T2 + T3; further validate this hypothesis, cell proliferation assays were con- P = 0.005). There was no signiﬁcant relevance between TEX19 levels ducted using siRNA-mediated knockdown TEX19 in OVCAR-3 and and other clinicopathological parameters, including age, distant me- A2780. The eﬃciency of TEX19 down-regulation was detected by tastasis, and histological grade (P = 0.407, P = 0.275, and P = 0.188, western blotting and qRT-PCR, and siRNA3 was selected for further respectively). The details are shown in Table 2. studies (Fig. 3A). CCK-8 assays showed that cell proliferation, compared

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Fig. 3. Effect of knockdown of TEX19 on proliferation of OC Cells. Note: (A) Down-regulation efficiency of TEX19 by three siRNAs in OVCAR-3 and A2780 was confirmed by western blotting and qRT-PCR. (B) The effect of knockdown of TEX19 on the viability of OVCAR-3 and A2780 was detected by CCK-8 proliferation assay. (C) The effect of knockdown of TEX19 on the colony formation ability of OVCAR-3 and A2780 was analyzed by plate clone formation assay. Blank: un-transfected cells; NC: scrambled siRNA-transfected cells; siTEX19: siRNA3-transfected cells. siTEX19 vs NC, *, P < 0.05; **, P < 0.01;***, P < 0.001. Abbreviations: TEX19, testis expressed 19; OC, ovarian cancer. with NC groups, was significantly suppressed in the TEX19-down- data demonstrated that TEX19 might play a crucial role in maintaining regulated group (P < 0.05; Fig. 3B). In plate clone formation assay, the proliferative capacity of OC cells. colonies formed in the TEX19-downregulated group were smaller and fewer than that in NC group (P < 0.05; Fig. 3C). Taken together, our

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Fig. 4. Effect of knockdown of TEX19 on invasion and migration of OC Cells. Note: (A) The effect of knockdown of TEX19 on the migration ability of OVCAR-3 and A2780 was verified by wound healing assay. (B) The effect of knockdown of TEX19 on the migration ability of OVCAR-3 and A2780 was detected by transwell migration assay. (C) The effect of knockdown of TEX19 on the invasion ability of OVCAR-3 and A2780 was analyzed by transwell invasion assay. Blank: un-transfected cells; NC: scrambled siRNA-transfected cells; siTEX19: siRNA3-transfected cells. siTEX19 vs NC, *, P < 0.05; **, P < 0.01;***, P < 0.001. Abbreviations: TEX19, testis expressed 19; OC, ovarian cancer.

3.5. Down-regulation of TEX19 inhibited migration and invasion of OC cells promoting migration and invasion of OC cells. To examine the inﬂuence of TEX19 on migration and invasion of OC cells, we performed wound Since the high level of TEX19 was correlated with deeper invasion healing assay and transwell assay. As shown in Fig. 4A, wound healing and lymph node involvement of OC, TEX19 might be necessary for assay showed that the cell-covered areas were signiﬁcantly decreased in

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Table 3 Potential HLA-A*0201 restricted CTL epitope peptides derived from TEX19.

Epitope Start End Length Sequence Percentile rank ANN IC50 (nM) SMM IC50 (nM) CombLib score

GT 44 52 9 GLDPHFVPT 3.6 165.87 288.48 0.000067 TL 32 40 9 TLAAAPEGL 3.5 201.62 309.82 0.0000611 GL 4 12 9 GLPWRFEEL 3.4 267.77 272.34 0.000266 YQ 16 24 9 YLYASWMYQ 3.4 342.19 418.9 0.0000586

Abbreviations: TEX19, testis expressed 19; HLA, human leukocyte antigen; CTL, cytotoxic T cell; ANN, artificial neural network; SMM, stabilized matrix method; CombLib, combinatorial peptide libraries. the TEX19-downregulated groups compared with that in NC groups to be a target for cancer immunotherapy. (P < 0.05). In addition, we found that the number of migrated cells in In the present study, we examined the level of TEX19 in OC tissue transwell assay without Matrigel were significantly reduced in the samples and statistically analyzed the association between clinical data TEX19-downregulated groups compared with that in NC groups of these samples and TEX19 protein levels. Our results indicated that (P < 0.05; Fig. 4B). Transwell assay with Matrigel indicated that the level of TEX19 in OC tissues was significantly higher than that in TEX19 knockdown significantly decreased the number of invaded cells adjacent normal tissues and had a significant correlation with TNM (P < 0.05; Fig. 4C). The data of this study supported the migration and stage, depth of invasion, and lymph node involvement, which was invasion promoting role of TEX19 in OC cells, revealing that TEX19 consistent with previous reports [28]. Furthermore, we detected the might accelerate the progression of OC metastasis. level of TEX19 in four OC cell lines and one ovarian epithelial cell line. The results showed that TEX19 levels of most OC cell lines were higher 3.6. Prediction of HLA-A*0201 restricted CTL epitopes based on TEX19 than normal. We selected two cell lines with top TEX19 levels to construct TEX19-downregulated models. In these models, proliferation, Based on the above experimental results, we considered that TEX19 metastasis, and invasion of OC cells were significantly declined. These is a potential tumor target. Thus, we used the IEDB database, a CTL results suggested that up-regulation of TEX19 levels may accelerate epitope prediction and analysis tool, to predict candidate CTL epitopes growth and progression of ovarian cancer. with binding to HLA-A*0201. The candidate peptides were chosen Additionally, some studies reported that TEX19 mRNA might be mainly based on IC50 (IC50 < 500 nM) (Table 3). To further predict expressed regionally as the tumor progresses, which explained why the possibility of HLA-A*0201 (PDB code: 3V5K) binding to the four relative TEX19 mRNA expression is low in some databases [34]. As we peptides, YQ, GL, TL, and GT, we performed computational analysis know, a high degree of heterogeneity is a critical characteristic of tu- through the Pepsite2 prediction platform. Consistent with previous mors, and so database specimens may also contain information derived predictions, these four peptides could combine with HLA-A*0201 stably from tumor regions that don't express TEX19 mRNA [46,47]. Even so, (P < 0.05; Fig. 5). The above results indicated that the four candidate the differential expression of TEX19 mRNA is associated with poor epitopes could be promising peptide vaccine to analyze further. prognosis in esophagus cancer, breast cancer, kidney cancer, gall- bladder cancer, uveal melanoma, and prostatic cancer, according to 3.7. The binding affinity of the candidates and interactions of the best some previous reports and gene expression profiling interactive analysis epitope peptide with HLA-A*0201 (GEPIA) (http://gepia.cancer-pku.cn)[34,48]. At present, immunotherapy has become one of the most important To confirm the predicted results, we further evaluated the ability of strategies to fight cancers [49]. Nevertheless, progress on im- the epitope peptides to bind to the HLA-A*0201 molecule in vitro using munotherapy for ovarian cancer is still limited [50]. Fortunately, T2 cell-binding assays. It was found that only the epitope peptide TL bioinformatic prediction tools have been optimized to analyze and showed a high affinity with HLA-A*0201 molecule, and the remainings design multi-epitope vaccines, and the feasibility and accuracy have were low-affinity peptides (Fig. 6A). Correspondingly, the fluorescence been confirmed by many studies [51,52]. In consequence, we utilized index of the TL treatment group was significantly high compared with IEDB database and Pepsite2 prediction platform to predict and screen the critical value (FI = 1.5), NC group, and other treatment groups preliminarily candidate epitopes derived from TEX19, and then ob- (P < 0.05; Fig. 6B). To investigate the specific interaction between TL tained four potential HLA-A*0201-restricted epitope-peptides, which and HLA-A*0201 molecules, we used the MOE docking program to could be used in further immunoassays to validate the prediction and obtain the most promising conformation and the binding packet of avoid false positives. Our data suggested that TL has the best affinity HLA-A*0201 with TL (Fig. 6C and D). The contact analysis of interac- with HLA-A*0201, which is consistent with the previous prediction tion between TL and HLA-A*0201 molecule is shown in Table 4. Thus, using pepsite2 platform. these data indicated that TL as a candidate for designing an anti-tumor Structurally, class I MHC molecule (MHC-I) consists of two amino epitope vaccine targeting TEX19 might have a promising prospect. acid chains, the α1 and α2 chains, which both contain four β-pleated sheets and one α-helix. The two domains jointly form the peptide- 4. Discussion binding groove, which is closed at both ends. The most binding peptides have a length of nine amino acids [53,54]. An antigenic peptide gen- Testis Expressed 19 (TEX19) is one of the mammalian specific genes erally contains two or more sites that bind to MHC-I molecules and are recently identified as a cancer-testis gene according to its published referred to as an anchor site. Combining the characteristics of a large expression profile [26–28,33,44]. Many germline genes were associated number of antigenic peptides, the most common anchor sites for an- with poor prognosis in cancers, so revealing function of these genes is tigen peptides bound to HLA-A*0201 are the second and ninth posi- imperative to comprehend the mechanisms of tumor initiation/pro- tions, and the anchor residues are Leu/Met and Leu/Val, respectively gression [45]. Feichtinger et al. first revealed that TEX19 mRNA is [54]. The TL (TLAAAPEGL) candidate epitope satisfies the above-de- present in multiple cancers and found a significant correlation between scribed conditions well, and we evaluated the binding of the second or TEX19 and OC through meta-analysis [28]. Additionally, Planells-Palop ninth anchor residues to HLA-A*0201 using different prediction et al. previously reported that that TEX19 is required for the pro- methods, as presented in Fig. 4 and Table 3. The electron analysis re- liferation and self-renewal of cancer cells [34]. Therefore, TEX19 is vealed that 8 binding bonds allow TL to exhibit considerable affinity for likely to play an essential role in the development of OC and is expected HLA-A*0201, and 7 amino acid residues of HLA-A*0201 are involved

8 Z. Xu, et al. Life Sciences 241 (2020) 117171

Fig. 5. Computational analysis of the docking possibility of HLA-A*0201 and the four peptides based on the PepSite2 web server. Note: The representative protein structure, a predicted peptide binding spot, and the top one matches ranked according to statistical signiﬁcance (p-value) for YQ (A), GL (B), TL (C), and GT (D). Abbreviations: HLA, human leukocyte antigen.

(Glu55, Arg97, Arg170, Glu58, Tyr159, Glu63, and Lys66). Among identified a dominant epitope TL (TLAAAPEGL) that can effectively them, Glu55 and Arg97 of HLA*0201 interacted strongly with Thr1 and bind to HLA-A*0201. These results demonstrated that the elevation of Leu9 of TL, respectively, both of which contained an ionic bond and a TEX19 promotes malignant phenotype of OC, and the potential peptide covalent bond. The study of the interaction between TL and HLA- TL derived from TEX19 may be used as an epitope vaccine for the A*0201 is a key step to further structural optimization and mutation treatment of OC. optimization in the future. The above results suggested that TL could be Supplementary data to this article can be found online at https:// a promising candidate to design and construct an anti-OC epitope doi.org/10.1016/j.lfs.2019.117171. vaccine. Therefore, it will be important to explore the effect of acti- vating T cells to kill OC cell. Declaration of competing interest

5. Conclusion The authors conﬁrm that there are no conﬂicts of interest.

To sum up, our study ﬁrstly found the protein level of TEX19 was Acknowledgements elevated in OC tissue samples and was signiﬁcantly associated with proliferation, metastasis, and invasion of OC cells. Furthermore, we This work was supported by National Natural Science Foundation of

9 Z. Xu, et al. Life Sciences 241 (2020) 117171

Fig. 6. The binding ability of the candidates and the prediction of interaction between TL and HLA-A*0201. Note: (A) The binding ability between the epitope peptides and HLA-A*0201 molecule was evaluated by T2 cell binding assay. (B) The affinity was compared by FI, which calculated by MFI of each group. (C) The interacting packet of HLA-A*0201 molecule with TL presented by 3D model. (D) Binding interaction of TL with the key residues of HLA-A*0201 molecule shown by 2D model. NC: β2-MG treatment group; TL vs NC, ****, P < 0.0001. Abbreviations: HLA, human leukocyte antigen; FI, fluorescein index; MFI, mean fluorescence intensity; β2–MG, β2-microglobulin.

Table 4 providing the space and equipment for conducting the experiments. Contacts between HLA-A*0201(PDB code:3V5K) and TL.

Type of Chain A Set A Chain B Set B Energy Distance References connection (kcal/mol) [1] B. F, F. J, S. I, S. RL, T. LA, J. A, Global cancer statistics, GLOBOCAN estimates of Ionic/hydrogen TL Thr1 3V5K-A Glu55 −20.834 2.852 incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. bond Clin. 68 (2018) (2018) 394–424. Ionic/hydrogen TL Leu9 3V5K-A Arg97 −25.973 2.733 [2] C. SB, B. F, S. ME, T. B, International patterns and trends in ovarian cancer in- bond cidence, overall and by histologic subtype, International journal of cancer 140 – Hydrogen bond TL Thr1 3V5K-A Arg170 −1.700 3.098 (2017) 2451 2460. Hydrogen bond TL Ala3 3V5K-A Glu58 −2.200 2.734 [3] I.J. Jacobs, U. Menon, Progress and challenges in screening for early detection of – Hydrogen bond TL Ala4 3V5K-A Glu58 −7.700 2.930 ovarian cancer, Mol. Cell. Proteomics 3 (2004) 355 366. [4] Y. A, L. ME, C. JR, M. HA, Breast and ovarian cancers: a survey and possible roles Hydrogen bond TL Pro6 3V5K-A Tyr159 −3.500 2.799 for the cell surface heparan sulfate proteoglycans, The journal of histochemistry and Hydrogen bond TL Glu7 3V5K-A Glu63 −5.300 3.029 cytochemistry : official journal of the Histochemistry Society 60 (2012) 9–21. − Hydrogen bond TL Glu7 3V5K-A Lys66 6.200 2.892 [5] M.G. Teneriello, R.C. Park, Early detection of ovarian cancer, Cancer 117 (2011) 4340–4342. Abbreviations: TEX19, testis expressed 19; HLA, human leukocyte antigen; [6] R. Nuttall, Canadian Cancer Statistics 2017, Canadian Cancer Society (2017). PDB, protein data bank. [7] A. Noone, N. Howlader, M. Krapcho, D. Miller, A. Brest, M. Yu, J. Ruhl, Z. Tatalovich, A. Mariotto, D. Lewis, SEER Cancer Statistics Review, National Cancer Institute, Bethesda, MD, 2018 1975-2015, based on November 2017 SEER China [Nos 81601370]; NSFC-Liaoning Joint Fund Key Program [No. data submission, posted to the SEER web site (April 2018) https://seer.cancer.gov/ U1608281]; Shenyang S&T Projects [19-109-4-09, 17-123-9-00]; and csr/1975_2015/. Double Hundred Program for Shenyang Scientific and Technological [8] D. Jelovac, D.K. Armstrong, Recent progress in the diagnosis and treatment of ovarian cancer, Ca A Cancer Journal for Clinicians 61 (2011) 183–203. Innovation Projects [Z18-4-020]. [9] A.M. Oza, A.D. Cook, J. Pfisterer, A. Embleton, J.A. Ledermann, E. Pujadelauraine, The authors would like to acknowledge the Key Laboratory of G. Kristensen, M.S. Carey, P. Beale, A. Cervantes, Standard chemotherapy with or Precision Diagnosis and Treatment of Gastrointestinal Tumors, Ministry without bevacizumab for women with newly diagnosed ovarian cancer (ICON7): of Education (China Medical University, Shenyang, China) for overall survival results of a phase 3 randomised trial, Lancet Oncol. 16 (2015) 928–936.

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