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TRIP13 promotes tumor growth and is associated with poor prognosis in colorectal cancer

TRIP13 promotes tumor growth and is associated with poor prognosis in colorectal cancer Colorectal cancer (CRC) is one of the most common neoplasms worldwide. However, the mechanisms underlying its development are still poorly understood. Thyroid hormone Receptor Interactor 13 (TRIP13) is a key mitosis regulator, and recent evidence has shown that it is an oncogene. Here, we report that TRIP13, which is overexpressed in CRC, is correlated with the CEA (carcino-embryonic antigen), CA19-9 (carbohydrate antigen 19-9) and pTNM (pathologic primary tumor, lymph nodes, distant metastasis) classification. Multivariate analyses showed that TRIP13 might serve as an independent prognostic marker of CRC. We also found that TRIP13 promoted CRC cell proliferation, invasion and migration in vitro and subcutaneous tumor formation in vivo. Furthermore, the potential mechanism underlying these effects involves the interaction of TRIP13 with a 14-3-3 protein, YWHAZ, which mediates G2-M transition and epithelial- mesenchymal transition (EMT). Together, these findings suggest that TRIP13 may be a potential biomarker and therapeutic target for CRC. Introduction spindle assembly checkpoint and chromosome synapsis . Colorectal cancer (CRC) is one of the most prevalent Previous reports have indicated that TRIP13 was over- 1 5–7 and fatal malignancies worldwide . In China, CRC is expressed in multiple neoplasms . Recently, one study among the top five cancers both in new diagnoses and in from Japan indicated that TRIP13 may act as an oncogene the leading cause of death . Although great progress in in colorectal cancer cells, but the mechanism was not surgery and chemotherapy has been achieved during the clarified . In this study, we used multiple methods to past few decades, the 5-year overall survival rate for stage demonstrate that TRIP13 can promote CRC cell pro- III CRC is 59.5% and only 8.1% for stage IV CRC patients . liferation, migration, and invasion in vitro and sub- Relapse and metastasis are the main causes of high cutaneous implanted tumor formation in vivo, and it mortality, but the underlying molecular mechanisms have predicted poor survival of CRC patients. The potential not been fully elucidated. mechanism underlying these effects involves TRIP13 Thyroid hormone Receptor Interactor 13 (TRIP13), has interaction with YWHAZ, a member of the 14-3-3 protein been found to play a key role in meiotic recombination, family, which mediates G2-M transition and epithelial- mesenchymal transition (EMT). Correspondence: Gewen Tan (zerogwtan@hotmail.com)or Results Hongqi Chen (hqchen08@163.com) or Zhigang Wang (surlab@hotmail.com) Expression of TRIP13 in human CRC tissues Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China To determine the expression profile of TRIP13 in CRC, State Key Laboratory of Medical Genomics, Shanghai Institutes for Biological we analyzed multiple CRC data sets available from Sciences, Chinese Academy of Sciences, Shanghai 200031, China These authors contributed equally: Nengquan Sheng, Li Yan, Kai Wu. Edited by G.-Q. Chen © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Sheng et al. Cell Death and Disease (2018) 9:402 Page 2 of 14 Fig. 1 TRIP13 is overexpressed in CRC and associated with poor prognosis. a Data set from Oncomine showing that TRIP13 is upregulated in tumor tissue compared to normal tissue. b TRIP13 mRNA is overexpressed in tumor tissue compared with normal tissue in 41 paired CRC patients from TCGA. n = 41, p < 0.001. c Western blotting of 45 paired tumor and adjacent normal tissues shows that TRIP13 is highly expressed in tumor tissue. The upper panel is the signal intensity of TRIP13 and GAPDH; the lower panel shows the quantified results. n = 45, p < 0.001. d TRIP13 IHC scores of the staining intensity level in representative tumor tissues. The bars are as indicated. e OS was compared between patients with high and low expression of TRIP13. p = 0.0001 Oncomine, and TRIP13 was found to be upregulated in Furthermore, to assess the protein level of TRIP13 in tumor tissue compared with normal tissue in these data CRC, western blotting was performed in 45 paired CRC sets (Fig. 1a from left to right: Graudens Colon , Hong and adjacent normal tissues, and we found that TRIP13 10 11 colorectal , Skrzypczak colorectal2 , Skrzypczak color- was significantly overexpressed in tumors compared with 11 12 13 ectal , Sabates Bellver colon , Gaedcke colorectal ,Ki the matched normal tissues (Fig. 1c, p < 0.001). Together, colon .) The accession numbers are GSE3964, GSE9348, these data indicate that TRIP13 is upregulated in CRC. GSE20916, GSE20196, GSE8671, GSE20842, and GSE6988. To validate the results, we examined the mRNA Increased TRIP13 expression contributes to poor prognosis level of TRIP13 in 41 pairs of CRC and corresponding in CRC patients normal tissues from TCGA (The Cancer Genome Atlas) To investigate the potential prognostic value of TRIP13 data set. The results also showed that TRIP13 was highly in CRC, immunohistochemistry was performed with 200 expressed in tumor tissue (Fig. 1b, p < 0.001). CRC formalin-fixed, paraffin-embedded (FFPE) tumor Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 3 of 14 Table 1 Comparison of clinicopathological profiles Table 2 Univariate and multivariate analysis of factors between low and high TRIP13 expression in CRC patients associated with overall survival in CRC patients Low High p Features Overall survival expression expression Univariate Multivariate (n = 89) (n = 111) p-value HR 95% CI p-value n % n % Age: ≥60 vs. <60 years 0.295 NA Age 0.294 Gender: male vs. female 0.276 NA <60 31 34.8 31 27.9 Tumor location: colon vs. 0.394 NA ≥60 58 65.2 80 72.1 rectal Gender 0.938 Differentiation: poor vs. 0.882 NA Female 38 42.7 48 43.2 moderate & well Male 51 57.3 63 56.8 3 Tumor size: ≥15 vs. <15 cm 0.063 NA Tumor location 0.709 pTNM: I–II vs. III–IV <0.001*** 3.391 2.291–5.019 <0.001*** Rectal 27 30.3 31 27.9 CEA: ≥5 vs. <5 ug/l <0.001*** 1.7 1.2–2.406 0.03* Colon 62 69.7 80 72.1 CA125: ≥35 vs. <35 U/ml 0.153 NA Tumor size 0.884 CA19-9: ≥37 vs. <37 U/ml 0.003** NS <15 cm 28 31.5 36 32.4 TRIP13: high vs. low <0.001*** 1.887 1.321–2.696 <0.001*** ≥15 cm 61 68.5 75 67.6 NA not adopted, NS not significant. *p < 0.05; **p < 0.01; ***p < 0.001 Differentiation 0.49 In addition, Cox proportional hazards regression ana- Poor 18 20.2 27 24.3 lyses (Table 2) demonstrated that TRIP13 was an inde- Moderate & well 71 79.8 84 75.7 pendent prognostic predictor for overall survival (HR = pTNM 0.005** 1.887, p < 0.001). Moreover, Kaplan-Meier analysis I-II 49 55.1 39 35.1 showed that patients with high TRIP13 expression III-IV 40 44.9 72 64.9 exhibited poorer survival than those with low expression (Fig. 1e, HR = 1.961, p = 0.0001). These data indicated CEA 0.045* that TRIP13 is significantly associated with CRC patient <5 μg/l 59 66.3 58 52.3 prognosis. ≥5 μg/l 30 33.7 53 47.7 Altered TRIP13 in CRC cells and its effect on tumorigenic CA125 0.378 properties in vitro <35 U/ml 66 74.2 76 68.5 To evaluate the biology function of TRIP13 in CRC ≥35 U/ml 23 25.8 35 31.5 cells, TRIP13 expression was knocked down with siRNAs CA19-9 0.043* and LV10-shRNA, and TRIP13 overexpression was induced with LV5-TRIP13. The transfection efficiency <37 U/ml 68 76.4 70 63.1 and TRIP13 protein expression level were validated by ≥37 U/ml 21 23.6 41 36.9 immunofluorescence and western blotting, respectively Chi-square test was performed, and p < 0.05 was considered statistically (Fig. 2a and Fig. 3a for TRIP13 overexpression, Fig. 2b–c significantly. *p < 0.05; **p < 0.01 and Fig. 4a for TRIP13 knockdown). (Fig. 2a and Fig. 3a pTNM pathological tumor, lymph node, metastasis classification, CEA carcino- embryonic antigen, CA125 carbohydrate antigen-125, CA19-9 carbohydrate for TRIP13 overexpression, Figs. 2b, c and Fig. 4a for antigen-19-9 TRIP13 knockdown). Simultaneously, we cloned the second siRNA sequence (si2) that targeted TRIP13 into an slides with complete clinicopathological characteristics LV10 vector that also encoded RFP. and follow-up data. These patients were divided into high Then, we examined the effect of TRIP13 overexpression and low groups according to TRIP13 expression using the and knockdown on the proliferative ability of cells using scoring system described in the Methods (Fig. 1d). We MTT and colony formation assays. The results showed found that high TRIP13 expression was significantly that overexpression of TRIP13 increased cell viability associated with advanced pTNM stage and higher CEA compared with the negative control (Fig. 3b), and this and CA19-9 expression (Table 1). promotive effect was validated by the colony formation Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 4 of 14 Fig. 2 Transfection efficiency was examined with an immunofluorescence assay. a IF was performed to validate the overexpression efficiency of TRIP13 in HCT116 and SW480 cells. Red indicates TRIP13, and blue indicates cell nuclei. The bar is 50 μm. b and c IF was performed to validate the knockdown efficiency of TRIP13 in SW480 and HCT116 cells. Red and green indicate TRIP13, and blue indicates cell nuclei. The bar is 50 μm assay, in which TRIP13 led to increased colony formation TRIP13 on cell migration and invasion. These assays (Fig. 3c). And knockdown of TRIP13 decreased cell via- showed that cells overexpressing TRIP13 have increased bility (Figs. 4b, c). Furthermore, transwell migration and migration and invasion ability compared with negative invasion assays were employed to assess the influence of control cells (Figs. 3d, e), while knockdown TRIP13 Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 5 of 14 Fig. 3 Upregulation of TRIP13 promotes tumorigenic properties in vitro. a Validation of the efficiency of TRIP13 expression. The upper panel is signal intensity; the lower panel is the quantified results. The values indicate the mean ± standard deviation. p < 0.01. b–e The effects of TRIP13 gain of function on in vitro proliferation (b, c), migration (d), and invasion (e). TRIP13 up-regulation significantly increased cell proliferation, migration and invasion abilities. The values indicate the mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001 induced the opposite effect (Figs. 4d, e). In addition, Figure 1A-D). Taken together, these data indicate that similar results with HCT116 and SW480 cells transduced TRIP13 promotes the proliferation, invasion and migra- with LV10-NC and LV10-shTRIP13 (Supplementary tion ability of CRC cells. Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 6 of 14 Fig. 4 Suppression of TRIP13 inhibits the oncogenic phenotype in vitro. a Confirmation of the efficiency of TRIP13 knockdown. The left panel is signal intensity, and the right panel is the quantified results. The values indicate the mean ± standard deviation. p < 0.05. b–e The effects of TRIP13 loss of function on in vitro proliferation (b, c), migration (d), and invasion (e). TRIP13 knockdown inhibits cell proliferation, migration and invasion abilities. The values indicate the mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001 Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 7 of 14 Fig. 5 Altered TRIP13 expression affects tumorigenicity in vivo. a Tumors in nude mice bearing HCT116 cells in different groups. The upper panel is TRIP13 overexpression and the negative control; the lower panel is TRIP13 knockdown and the negative control. Each group contains six mice. b Statistical analysis of tumor volume in different groups. The left panel shows the overexpression and control groups, and the right panel shows the knockdown and control groups. The values indicate the mean ± standard deviation. n = 6/group. **p < 0.01; ***p < 0.001. c Statistical analysis of tumor weight in different groups. The left panel shows the overexpression and control groups, and the right panel shows the knockdown and control groups. The values indicate the mean ± standard deviation. n = 6/group. ***p < 0.001. d Representative staining images of TRIP13 overexpressing tumors. The bars are as indicated. e Representative staining images of TRIP13 knockdown tumors. The bars are as indicated Altered TRIP13 expression affects tumorigenicity in vivo larger tumors than those injected with LV5-NC- The above studies demonstrated that TRIP13 promotes transfected HCT116 cells (Fig. 5a, top panel; Fig. 5b, left the tumorigenic potential of CRC in vitro. Next, we panel). Knockdown of TRIP13 had the opposite effect, explored the effect of TRIP13 on tumorigenesis in vivo and the subcutaneous tumors formed from LV10- using a xenograft model. Nude mice injected with LV5- shTRIP13-infected HCT116 cells were smaller than TRIP13-infected HCT116 cells were found to have much those formed from LV10-NC-infected HCT116 cells Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 8 of 14 (Fig. 5a, bottom panel; Fig. 5b, right panel). These results demonstrated that 14-3-3 proteins play a key role in cell were validated by the tumor weights (Fig. 5c). cycle, apoptosis, and EMT . Collectively, these data Then, H&E staining was used to confirm the tumori- provide strong evidence that TRIP13 plays an important genic characteristics of these subcutaneous tumors part in cell cycle regulation pathways. (Figs. 5d, e, bottom panel), and immunohistochemistry staining was performed with those confirmed cancer tis- TRIP13 modulates EMT in an YWHAZ-regulated way sues. The results showed that TRIP13 was high expressed As YWHAZ interacts with TRIP13 and mediates EMT, in TRIP13 overexpressing tumors and low expressed in TRIP13 promotes EMT, we proposed that TRIP13 may TRIP13 knockdown tumors (Figs. 5d, e, the second panel). mediate EMT dependent on interacting with YWHAZ. In addition, Ki-67 immunohistochemistry staining was To assess this hypothesis, three different siRNAs (si1, si2, used to assess the proliferative index of these tumors. The si3) targeting YWHAZ were applied to transfect TRIP13 results showed that upregulation of TRIP13 increased the overexpressing cells, the known molecular markers of proliferative ability of cells, but downregulation of EMT, TRIP13 and YWHAZ were examined by western TRIP13 suppressed the cell proliferative ability (Figs. 5d, blotting. The results showed that knockdown of YWHAZ e, the first panel). Together, these data suggest that neutralized the up-regulation effect of N-cadherin, β- TRIP13 may promote tumorigenesis in vivo. catenin, snail, and the down-regulation effect of E- cadherin (Fig. 7a). And then down-regulation of TRIP13 is correlated with EMT in CRC cells YWHAZ induced inhibition of migration and invasion EMT is recognized as an important process during ability in both HCT116 and SW480 cells (Fig. 7b,c). carcinoma progression . To determine whether TRIP13 Moreover, the expression of YWHAZ in xenograft tumors has a critical role in the CRC EMT process, we examined was examined by immunohistochemistry, the results changes in known molecular markers of EMT using showed that YWHAZ was high expressed in TRIP13 western blotting in both HCT116 and SW480 cells. The overexpressing tumors and low expressed in TRIP13 results showed that upregulation of TRIP13 increased the knockdown tumors (Figs. 5d, e, the third panel). Fur- expression of N-cadherin, β-catenin and snail but sup- thermore, we evaluated the expression of YWHAZ in the pressed E-cadherin expression (Fig. 6a). In contrast, same 200 CRC FFPE tumor slices, the representative knockdown of TRIP13 suppressed N-cadherin, β-catenin staining scores were shown in Fig. 7d. Spearman corre- and snail and increased E-cadherin expression (Fig. 6b). lation analyses was conducted, and the result showed that Cells transduced with LV10-NC and LV10-shTRIP13 TRIP13 and YWHAZ were positively related (Fig. 7e, r = showed similar results (Supplementary Figure 1E). These 0.532, p < 0.001). These data together indicate that data demonstrated that TRIP13 may promote CRC pro- TRIP13 modulates EMT in an YWHAZ-dependent way. gression by inducing EMT. Discussion TRIP13 interacts with 14-3-3 and modulates cell cycle In the past few decades, a series of biomarkers have To explore the molecular mechanism of TRIP13 in been identified as having prognostic value and therapeutic CRC, we sought to identify potential TRIP13-interacting significance in CRC. However, the overall survival of late proteins. Flag-tagged TRIP13 was expressed in HCT116 stage CRC patients remains poor, and our understanding cells, stained and immunoblotted, and then quantified of the mechanisms of this disease is still inadequate. In using nano-LC-MS/MS (Fig. 6c). A large number of this study, we demonstrated that TRIP13 is a potential proteins were found to interact with TRIP13, and the CRC biomarker. genes encoding these proteins were annotated (Supple- TRIP13, a member of the AAA + ATPase super-family, mentary Excel 1). Then, gene-ontology (GO) Enrichment is located at 5p15.33 and encodes a protein with 432 Analysis was performed to annotate the biological pro- amino acids. Studies have shown that TRIP13 is a key cesses in which these genes are involved (Supplementary regulator of meiotic recombination and the spindle Excel 2). We found that these genes were enriched in cell assembly checkpoint because TRIP13 catalyzes the con- cycle process (Fig. 6d, Supplementary Excel 2). Further- version of C-Mad2 to inactive O-Mad2, with the help of comet more, Kyoto Encyclopedia of Genes and Genomes adapter protein p31 to recruit TRIP13 to HORMA 17,18 (KEGG) pathway analysis validated that these genes par- domain-closure motif complexes . Despite playing a ticipated in cell cycle process (Supplementary Excel 3). In key role in meiotic regulation, overexpression or ampli- addition, STRING database shows a clear interaction fication of TRIP13 has been found in multiple human 19,20 network between TRIP13-interacting genes and other cancers . In our study, TRIP13 was found to be highly DNA replication proteins (Fig. 6e). The TRIP13- expressed in CRC tissues compared with adjacent normal interacting genes YWHAQ and YWHAZ are members tissues, and this result was validated by the analysis of of the 14-3-3 superfamily. A previous study has CRC data sets from Oncomine and TCGA. TRIP13 was Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 9 of 14 Fig. 6 TRIP13 regulates G2-M transition and EMT. a, b Western blotting was performed in HCT116 and SW480 cells to determine the change in EMT markers expression upon gain/loss function of TRIP13. The signal intensity and quantitative analysis are as shown. The values indicate the mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. c MS analysis of TRIP13-associated proteins. Cell lysate extract from Flag-tagged TRIP13- expressing or control was subjected to IP, and then, the IP complex was subjected to MS analysis. d GO analysis was performed to determine the biological processes in which TRIP13-interacting genes are involved. e The interaction network of TRIP13-interacting genes from STRING database Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 10 of 14 Fig. 7 TRIP13 mediates EMT by interacting with YWHAZ. a Western blotting of EMT markers, TRIP13 and YWHAZ were performed in TRIP13 overexpressing CRC cells which transfected with siRNAs targeting YWHAZ, to determine whether TRIP13 mediates EMT in a YWHAZ dependent way. b, c The effect of knockdown of YWHAZ in TRIP13 overexpressing CRC cells on migration (b), and invasion (c). d YWHAZ IHC scores of the staining intensity level in representative tumor tissues. The bars are as indicated. e Spearman correlation coefficient plots for TRIP13 and YWHAZ. r = 0.532, 95%CI: (0.421–0.627), p < 0.001 Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 11 of 14 first reported as an oncogene in 2014 in head and neck proteins that interact with TRIP13, and then, GO and cancer, and several subsequent studies revealed that KEGG analysis were applied to explore the biological 5–8 TRIP13 plays an oncogenic role in other neoplasms . function and pathway of the genes encoding these pro- Kurita and his colleagues have demonstrated that TRIP13 teins. The results indicated that TRIP13-interacting genes involves in CRC cell proliferation and invasion and may are involved in G2-M cell cycle transition. Cell cycle has be a potential target for CRC treatment , but they only four sequential phases, G1 (preparation for DNA synth- confirmed the oncogenic role of TRIP13 in vitro, and the esis), S (DNA replication), G2 (preparation for mitosis), mRNA level of TRIP13 was analyzed between normal and and M (mitosis), and each phase is highly controlled and tumor tissues from unpaired patients. Here, we demon- regulated by checkpoints, which aim to maintain genomic strated that TRIP13 promotes HCT116 and SW480 cells integrity by repairing damaged DNA before entering proliferation, migration and invasion, as well as tumor- mitosis. If the damaged DNA cannot be repaired cor- igenicity in vivo. Furthermore, we analyzed TRIP13 with rectly, cell cycle will be arrested . Checkpoints at G1-S clinicopathological characteristics in 200 CRC patients, transition and G2-M transition are the most important and found that TRIP13 was significantly associated with cell cycle checkpoints . However, during cancer cell pTNM stage, CEA and CA19-9. pTNM can be considered evolution, this mechanism is abolished due to mutated as the gold standard for cancer treatment and prognostic checkpoints, and then, damaged DNA can enter mitosis, predictor , CEA and CA19-9 is used to detect relapse and which leads to uncontrolled proliferation and a malignant 22 29 follow response to therapy of CRC . Moreover, multi- phenotype . Our study demonstrated that TRIP13 variate analyses showed that TRIP13 can predict overall interacts with YWHAQ and YWHAZ, which are mem- survival of CRC as well as pTNM and CEA. Therefore, bers of the 14-3-3 protein superfamily. YWHAZ, also TRIP13 may serve as a promising biomarker of CRC. named 14-3-3ζ, plays key roles in cell cycle and EMT , The mechanism by which TRIP13 contributes to recent researches indicated that YWHAZ involves in tumors is still poorly understood. In multiple myeloma, tumor progression and could be a prognostic marker for 30,31 researchers found that TRIP13 induces Mad2 degradation kinds of cancers . So we hypothesis that TRIP13 through the Akt pathway and abrogates spindle check- mediates EMT dependent on interacting with YWHAZ, point . As one of the mitotic checkpoint complexes, Mad2 knockdown of YWHAZ in TRIP13 overexpressing CRC is critical for chromosome segregation . Degradation of cells and western blotting of E-cadherin, N-cadherin, β- Mad2 leads to chromosome mis-segregation during catenin and snail were performed, the results showed that mitosis, which ultimately contributes to cancer develop- the up-regulation effect of N-cadherin, β-catenin, snail, ment and chemotherapy resistance . Zhou and his col- and the down-regulation effect of E-cadherin induced by leagues confirmed that the C-MYC/TRIP13/PUMA axis TRIP13 were rescued by YWHAZ knockdown, further- regulates chronic lymphocytic leukemia . PUMA, a pro- more, we found that TRIP13 and YWHAZ were positively apoptotic protein, was reported to be involved in p53- related. This data together confirmed our hypothesis, mediated apoptosis . However, in this study, researchers TRIP13 may promote CRC progression by interacting found that PUMA mediates cell apoptosis in a TRIP13- with YWHAZ to regulate EMT. dependent manner. As the first research indicated that In sum, TRIP13 promotes CRC cell progression in vivo TRIP13 acts as an oncogene, Banerjee and his colleagues and in vitro and indicates poor CRC patient survival. The utilized mass spectrometry and Co-IP to explore how underlying mechanism involves regulation of G2-M TRIP13 promotes head and neck cancer progression, and transition and EMT through interacting with YWHAZ. they found that TRIP13 promotes non-homologous end Our study suggests that TRIP13 is a potential biomarker joining (NHEJ) and induces chemoresistance . Double- for CRC patients and might further assist in therapeutic strand breaks are the most lethal type of DNA damage, decisions regarding CRC treatment. and they are primarily repaired through homologous recombination (HR) or NHEJ . NHEJ promotes cancer Materials and methods and chromosome instability because it is often inaccurate. Data resource It is well recognized that EMT gives rise to the dis- The analyses of TRIP13 in CRC was performed using semination of tumor cells from primary sites, which Oncomine (www.oncomine.org), which comprised of 7 polarized epithelial tumor cells acquired mesenchymal groups. The number of tissues included was 12 normal and cell phenotype and enhanced migratory capacity and 18 tissues in group 1, 12 normal and 70 tumor tissues in invasiveness . So we examined the effect of TRIP13 on group 2, 10 normal and 5 tumor tissues in group 3, 24 expression of critical EMT markers, E-cadherin, N-cad- normal and 45 tumor tissues in group 4, 32 normal and 32 herin, β-catenin and snail, and found that TRIP13 pro- tumor tissues in group 5, 65 normal and 65 tumor tissues motes EMT process. In order to clarify the underlying in group 6, 38 normal and 50 tumor tissues in group 7. We mechanism, Mass spectrometry was performed to analyze also downloaded level-3 RNAseq expression data of 41 Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 12 of 14 tumor and normal paired COAD from TCGA database, Western blot the TRIP13 HTseq-FPKM in tumor and normal samples Protein was prepared by lysing cells in radio immuno- were abstracted and statistical analyses was used by t-test. precipitation buffer containing phosphatase and protease inhibitors (Beyotime Biotechnology, Nantong, China) and Cell culture quantified using a BCA protein assay kit (Beyotime Bio- The human CRC cell lines HCT116 and SW480 were technology, Nantong, China). In brief, 50 µg of protein purchased from Cell Bank of Chinese Academy of Sci- was resolved in 8–15% Tris-SDS-PAGE gels and trans- ences (Shanghai, China) and cultured in Dulbecco’s ferred to PVDF membranes (Millipore, Billerica, USA). modified Eagle’s medium (DMEM) (Gibco, Carlsbad, The membranes were probed with a primary anti-human USA) supplemented with 10% fetal bovine serum (FBS) TRIP13 rabbit antibody (Proteintech, Chicago, USA) at a (Gibco, Carlsbad, USA) and 1% penicillin/streptomycin dilution of 1:1000 and incubated with HRP-conjugated (Gibco, Carlsbad, USA) in a humidified atmosphere of 5% Affinipure goat anti-rabbit secondary antibody (Pro- CO at 37 °C. teintech, Chicago, USA). Immunoreactive proteins were detected using ECL (Millipore, Billerica, USA) and a Bio- Tissue samples and clinicopathological information rad ChemoDoc MP System. The intensity of lanes was CRC tissues and paired normal tissues were obtained assessed using ImageJ (NIH, USA) from 45 patients who underwent surgical resection in the Department of General Surgery, Shanghai Jiao Tong Uni- Cell transfection and lentivirus infection versity Affiliated Sixth People’s Hospital. The fresh samples We successfully established stable TRIP13- were transported in liquid nitrogen and stored in −80 °C overexpressing HCT116 and SW480 cell lines via infec- until protein extraction. FFPE tumor blocks from 200 CRC tion with LV5-TRIP13 or LV5-NC lentivirus that also patients between January 2010 and January 2012 were encoded GFP. And we constructed two different siRNAs obtained from the Department of Pathology. A complete (si1, si2) and then applied them to transfect HCT116 and follow-up was conducted every 6 months until July 2017. SW480 cells using Lipofectamine 2000. Simultaneously, This study was approved by the Ethics Committee of we cloned the second siRNA sequence (si2) that targeted Shanghai Jiao Tong University Affiliated Sixth People’s TRIP13 into an LV10 vector that also encoded RFP. Hospital, and all patients provided informed consent. YWHAZ was knocked down with siRNAs. All of these constructs were purchased from GenePharma (Shanghai, Immunohistochemistry staining and scoring China), and transduction was performed according to the FFPE tumor blocks were cut into 5-μm-thick sections specific manufacturer’s instructions. and mounted on Premiere microscope slides, and then, the slides were heated at 60 °C for 40 min. Depar- Cell viability and colony formation assay affinization and rehydration was performed as previously Cell viability was assessed using an MTT Cell Pro- described . Antigen retrieval was performed with 10 nM liferation and Cytotoxicity Detection Kit (KeyGEN Bio- citrate antigen retrieval solution (Sangon Biotech, TECH, Nanjing, China). Colony formation ability was Shanghai, China) at 95 °C for 10 min. After non-specific assessed on 6 or 12-well plates for 10–14 days, and interactions were blocked with 3% goat serum, TRIP13 colonies were fixed with methanol and then stained with rabbit polyclonal antibody (Abcam, Cambridge, USA) was crystal violet. used at a dilution of 1:150, YWHAZ rabbit polyclonal antibody (Proteintech, Chicago, USA) was used at a Transwell migration and invasion assay dilution of 1:500, Ki-67 rabbit polyclonal antibody (Pro- Transwell assays were performed with 8-μm poly- teintech, Chicago, USA) with a dilution of 1:400, then carbonate transwell filters (Coring, Cambridge, USA). incubated with the slides overnight at 4 °C. Five random Briefly, 3–5×10 cells were seeded in the upper chamber fields in each slide were selected and evaluated indepen- without FBS, and the lower chamber was filled with 600 µl dently by two pathologists. A semi-quantitative scoring of DMEM containing 10% FBS. After 24–48 h of incu- system was used to evaluate the staining results basing on bation, cells on the lower membrane were set in 4% the percentage and intensity of positively stained cells. paraformaldehyde and stained with crystal violet. Then, 5 The intensity was scored as follows: 0-negative, 1-weak, 2- random fields were selected and photographed. moderate, and 3-strong. The frequency of positively stained cells was defined as: 0-less than 5%, 1–5 to 25%, Immunofluorescence assay 2–26 to 50%, 3–51 to 75%, and 4-greater than 75%. For After the indicated tumor cells crawled on the slide, statistical analysis, scores of 0 to 7 were considered low treatmented with siRNA or lentivirus, the CRC cells were expression, and scores of 8 to 12 were considered high fixed in 4% paraformaldehyde for 15 min. Then, the slides expression. were subjected to 0.2%Triton® X–100 (Sigma Aldrich, Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 13 of 14 St. Louis, USA) for 20 min and washed with PBST. serum-free DMEM (Gibco, Carlsbad, USA), and a total of Blocking was carried out in 5% bovine serum, and then 5×10 cells were injected subcutaneously into the right samples were incubated with anti-TRIP13 antibody flank of mice. The length and width of the resulting (Proteintech, Chicago, USA) at a dilution of 1:50 over- tumors were measured every 3 days starting 6 days after night at 4 °C. Then, the cells were incubated with Alexa- injection. Tumor volume was calculated using the formula labeled secondary antibodies (anti-rabbit IgG (H + L), F (length × width )/2. After 3 weeks, mice were sacrificed, (ab’)2 fragment (Alexa Fluor® 488 Conjugate)/anti-rabbit and the tumors were collected and weighed. The experi- IgG (H + L), F(ab’)2 fragment (Alexa Fluor® 594 Con- mental procedures were approved by the Ethical Com- jugate) 1:1000, Cell Signaling Technology, Danvers, MA, mittee of Shanghai Jiao Tong University Affiliated Sixth USA) for 30 min at room temperature. Nuclei were People’s Hospital. stained with DAPI (1:5000, Cell Signaling Technology, Danvers, USA). Following three final rinses with PBST, Statistical analysis the cells were imaged with a fluorescence microscope The data are presented as the mean ± standard deviation (Olympus, Japan). of at least three independent experiments. Difference between variables were assessed with a two-tailed t-test or Co-immunoprecipitation and peptide preparation χ analysis. The effect of clinical variables on patient HCT116-Flag-TRIP13 cells were lysed (CST, Danvers, survival was analyzed using Cox proportional hazards USA), and the protein was isolated. TRIP13 complex regression analyses, and Kaplan-Meier survival analysis proteins were purified with anti-Flag M2 magnetic was used to compare CRC patient survival with the log- beads (Sigma-Aldrich, St. Louis, USA). The co- rank test. For correlation, we used Spearman correlation. immunoprecipitation assay procedure was performed p < 0.05 was considered statistically significant. according to the manufacturer’s instructions. Then, the Acknowledgements TRIP13 complex components were eluted in blue loading This work was supported by Shanghai Municipal Education Commission- buffer (CST,Danvers,USA) and SDS-PAGE wasperformed Gaofeng Clinical Medicine Grant Support (no.20172023), the Collaborative Innovation Center for Translational Medicine at Shanghai Jiao Tong University (mini-PROTEAN precast gels, Bio-Rad). The protein lanes School of Medicine (no.TM201519), Shanghai Pujiang Program were visualized with Coomassie brilliant blue staining. (no.16PJ1408200), Natural Science Foundation of Shanghai (no.16ZR1449600), Peptide preparation for mass spectrometric analysis was National Natural Science Foundation of China (no.81602689). performed according to a previously described process . Author details Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth Mass spectrometry and peptide data analysis People’s Hospital, Shanghai 200233, China. State Key Laboratory of Medical The prepared peptides were analyzed using nano-LC- Genomics, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China MS/MS. LC separations were performed on an Easy nano LC system (Thermo Scientific, Bremen, Germany). Eluted Conflict of interest peptides were directly analyzed using tandem mass The authors declare that they have no conflict of interest. spectrometry (MS/MS) on an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Bremen, Ger- Publisher's note many) equipped with a nano-electrospray ion source. Springer Nature remains neutral with regard to jurisdictional claims in Proteome Discoverer 2.0 (Thermo Scientific) software was published maps and institutional affiliations. used to analyze the raw Xcalibur file generated from the Supplementary Information accompanies this paper at https://doi.org/ mass spectrometer. The SEQUEST search algorithm was 10.1038/s41419-018-0434-z. used to search unique peptides against a composite database comprising all the UniProt Homo sapiens data- Received: 12 October 2017 Revised: 10 February 2018 Accepted: 22 February 2018 bases (10/2016, 34361 entries). The minimum cutoff for peptide length was set at 7 amino acids, and the max- imum permissible missed cleavage was set at 2. The positive proteins were identified with a local FDR < 1% and at least one unique peptide per protein. References 1. Siegel,R.L.etal. Colorectal cancer statistics,2017. CA 67,177–193 (2017). 2. Chen, W. et al. Cancer statistics in China, 2015. CA 66,115–132 (2016). Tumor formation in nude mice 3. Brenner, H., Kloor, M. & Pox, C. P. Colorectal cancer. Lancet 383,1490–1502 Female BALB/c nude mice (5 weeks old) were bred in a (2014). 4. Vader, G. Pch2(TRIP13): controlling cell division through regulation of HORMA specific pathogen-free animal faculty and monitored every domains. 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Involvement of miR-451 in resistance to paclitaxel by reg- 17. Ye, Q. et al. TRIP13 is a protein-remodeling AAA+ATPase that catalyzes MAD2 ulating YWHAZ in breast cancer. Cell Death Dis. 8, e3071 (2017). conformation switching. eLife 4, e07367 (2015). 32. Tan, G. et al. Combination therapy of oncolytic herpes simplex virus HF10 and 18. Marks,D.H.etal. Mad2 Overexpression uncovers acriticalrolefor TRIP13 in bevacizumab against experimental model of human breast carcinoma mitotic exit. Cell Rep. 19,1832–1845 (2017). xenograft. Int. J. Cancer 136,1718–1730 (2015). 19. Carter, S. L., Eklund, A. C., Kohane, I. S., Harris, L. N. & Szallasi, Z. A signature of 33. Niessen, S., McLeod, I. & Yates, J. R. III. Silver staining, digestion, and extraction chromosomal instability inferred from gene expression profilespredictsclinical of peptides from an acrylamide gel for MS analysis. CSH Protoc. 7, pdb. outcome in multiple human cancers. Nat. Genet. 38,1043–1048 (2006). prot4661 (2006). 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TRIP13 promotes tumor growth and is associated with poor prognosis in colorectal cancer

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Copyright © 2018 by The Author(s)
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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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Abstract

Colorectal cancer (CRC) is one of the most common neoplasms worldwide. However, the mechanisms underlying its development are still poorly understood. Thyroid hormone Receptor Interactor 13 (TRIP13) is a key mitosis regulator, and recent evidence has shown that it is an oncogene. Here, we report that TRIP13, which is overexpressed in CRC, is correlated with the CEA (carcino-embryonic antigen), CA19-9 (carbohydrate antigen 19-9) and pTNM (pathologic primary tumor, lymph nodes, distant metastasis) classification. Multivariate analyses showed that TRIP13 might serve as an independent prognostic marker of CRC. We also found that TRIP13 promoted CRC cell proliferation, invasion and migration in vitro and subcutaneous tumor formation in vivo. Furthermore, the potential mechanism underlying these effects involves the interaction of TRIP13 with a 14-3-3 protein, YWHAZ, which mediates G2-M transition and epithelial- mesenchymal transition (EMT). Together, these findings suggest that TRIP13 may be a potential biomarker and therapeutic target for CRC. Introduction spindle assembly checkpoint and chromosome synapsis . Colorectal cancer (CRC) is one of the most prevalent Previous reports have indicated that TRIP13 was over- 1 5–7 and fatal malignancies worldwide . In China, CRC is expressed in multiple neoplasms . Recently, one study among the top five cancers both in new diagnoses and in from Japan indicated that TRIP13 may act as an oncogene the leading cause of death . Although great progress in in colorectal cancer cells, but the mechanism was not surgery and chemotherapy has been achieved during the clarified . In this study, we used multiple methods to past few decades, the 5-year overall survival rate for stage demonstrate that TRIP13 can promote CRC cell pro- III CRC is 59.5% and only 8.1% for stage IV CRC patients . liferation, migration, and invasion in vitro and sub- Relapse and metastasis are the main causes of high cutaneous implanted tumor formation in vivo, and it mortality, but the underlying molecular mechanisms have predicted poor survival of CRC patients. The potential not been fully elucidated. mechanism underlying these effects involves TRIP13 Thyroid hormone Receptor Interactor 13 (TRIP13), has interaction with YWHAZ, a member of the 14-3-3 protein been found to play a key role in meiotic recombination, family, which mediates G2-M transition and epithelial- mesenchymal transition (EMT). Correspondence: Gewen Tan (zerogwtan@hotmail.com)or Results Hongqi Chen (hqchen08@163.com) or Zhigang Wang (surlab@hotmail.com) Expression of TRIP13 in human CRC tissues Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China To determine the expression profile of TRIP13 in CRC, State Key Laboratory of Medical Genomics, Shanghai Institutes for Biological we analyzed multiple CRC data sets available from Sciences, Chinese Academy of Sciences, Shanghai 200031, China These authors contributed equally: Nengquan Sheng, Li Yan, Kai Wu. Edited by G.-Q. Chen © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Sheng et al. Cell Death and Disease (2018) 9:402 Page 2 of 14 Fig. 1 TRIP13 is overexpressed in CRC and associated with poor prognosis. a Data set from Oncomine showing that TRIP13 is upregulated in tumor tissue compared to normal tissue. b TRIP13 mRNA is overexpressed in tumor tissue compared with normal tissue in 41 paired CRC patients from TCGA. n = 41, p < 0.001. c Western blotting of 45 paired tumor and adjacent normal tissues shows that TRIP13 is highly expressed in tumor tissue. The upper panel is the signal intensity of TRIP13 and GAPDH; the lower panel shows the quantified results. n = 45, p < 0.001. d TRIP13 IHC scores of the staining intensity level in representative tumor tissues. The bars are as indicated. e OS was compared between patients with high and low expression of TRIP13. p = 0.0001 Oncomine, and TRIP13 was found to be upregulated in Furthermore, to assess the protein level of TRIP13 in tumor tissue compared with normal tissue in these data CRC, western blotting was performed in 45 paired CRC sets (Fig. 1a from left to right: Graudens Colon , Hong and adjacent normal tissues, and we found that TRIP13 10 11 colorectal , Skrzypczak colorectal2 , Skrzypczak color- was significantly overexpressed in tumors compared with 11 12 13 ectal , Sabates Bellver colon , Gaedcke colorectal ,Ki the matched normal tissues (Fig. 1c, p < 0.001). Together, colon .) The accession numbers are GSE3964, GSE9348, these data indicate that TRIP13 is upregulated in CRC. GSE20916, GSE20196, GSE8671, GSE20842, and GSE6988. To validate the results, we examined the mRNA Increased TRIP13 expression contributes to poor prognosis level of TRIP13 in 41 pairs of CRC and corresponding in CRC patients normal tissues from TCGA (The Cancer Genome Atlas) To investigate the potential prognostic value of TRIP13 data set. The results also showed that TRIP13 was highly in CRC, immunohistochemistry was performed with 200 expressed in tumor tissue (Fig. 1b, p < 0.001). CRC formalin-fixed, paraffin-embedded (FFPE) tumor Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 3 of 14 Table 1 Comparison of clinicopathological profiles Table 2 Univariate and multivariate analysis of factors between low and high TRIP13 expression in CRC patients associated with overall survival in CRC patients Low High p Features Overall survival expression expression Univariate Multivariate (n = 89) (n = 111) p-value HR 95% CI p-value n % n % Age: ≥60 vs. <60 years 0.295 NA Age 0.294 Gender: male vs. female 0.276 NA <60 31 34.8 31 27.9 Tumor location: colon vs. 0.394 NA ≥60 58 65.2 80 72.1 rectal Gender 0.938 Differentiation: poor vs. 0.882 NA Female 38 42.7 48 43.2 moderate & well Male 51 57.3 63 56.8 3 Tumor size: ≥15 vs. <15 cm 0.063 NA Tumor location 0.709 pTNM: I–II vs. III–IV <0.001*** 3.391 2.291–5.019 <0.001*** Rectal 27 30.3 31 27.9 CEA: ≥5 vs. <5 ug/l <0.001*** 1.7 1.2–2.406 0.03* Colon 62 69.7 80 72.1 CA125: ≥35 vs. <35 U/ml 0.153 NA Tumor size 0.884 CA19-9: ≥37 vs. <37 U/ml 0.003** NS <15 cm 28 31.5 36 32.4 TRIP13: high vs. low <0.001*** 1.887 1.321–2.696 <0.001*** ≥15 cm 61 68.5 75 67.6 NA not adopted, NS not significant. *p < 0.05; **p < 0.01; ***p < 0.001 Differentiation 0.49 In addition, Cox proportional hazards regression ana- Poor 18 20.2 27 24.3 lyses (Table 2) demonstrated that TRIP13 was an inde- Moderate & well 71 79.8 84 75.7 pendent prognostic predictor for overall survival (HR = pTNM 0.005** 1.887, p < 0.001). Moreover, Kaplan-Meier analysis I-II 49 55.1 39 35.1 showed that patients with high TRIP13 expression III-IV 40 44.9 72 64.9 exhibited poorer survival than those with low expression (Fig. 1e, HR = 1.961, p = 0.0001). These data indicated CEA 0.045* that TRIP13 is significantly associated with CRC patient <5 μg/l 59 66.3 58 52.3 prognosis. ≥5 μg/l 30 33.7 53 47.7 Altered TRIP13 in CRC cells and its effect on tumorigenic CA125 0.378 properties in vitro <35 U/ml 66 74.2 76 68.5 To evaluate the biology function of TRIP13 in CRC ≥35 U/ml 23 25.8 35 31.5 cells, TRIP13 expression was knocked down with siRNAs CA19-9 0.043* and LV10-shRNA, and TRIP13 overexpression was induced with LV5-TRIP13. The transfection efficiency <37 U/ml 68 76.4 70 63.1 and TRIP13 protein expression level were validated by ≥37 U/ml 21 23.6 41 36.9 immunofluorescence and western blotting, respectively Chi-square test was performed, and p < 0.05 was considered statistically (Fig. 2a and Fig. 3a for TRIP13 overexpression, Fig. 2b–c significantly. *p < 0.05; **p < 0.01 and Fig. 4a for TRIP13 knockdown). (Fig. 2a and Fig. 3a pTNM pathological tumor, lymph node, metastasis classification, CEA carcino- embryonic antigen, CA125 carbohydrate antigen-125, CA19-9 carbohydrate for TRIP13 overexpression, Figs. 2b, c and Fig. 4a for antigen-19-9 TRIP13 knockdown). Simultaneously, we cloned the second siRNA sequence (si2) that targeted TRIP13 into an slides with complete clinicopathological characteristics LV10 vector that also encoded RFP. and follow-up data. These patients were divided into high Then, we examined the effect of TRIP13 overexpression and low groups according to TRIP13 expression using the and knockdown on the proliferative ability of cells using scoring system described in the Methods (Fig. 1d). We MTT and colony formation assays. The results showed found that high TRIP13 expression was significantly that overexpression of TRIP13 increased cell viability associated with advanced pTNM stage and higher CEA compared with the negative control (Fig. 3b), and this and CA19-9 expression (Table 1). promotive effect was validated by the colony formation Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 4 of 14 Fig. 2 Transfection efficiency was examined with an immunofluorescence assay. a IF was performed to validate the overexpression efficiency of TRIP13 in HCT116 and SW480 cells. Red indicates TRIP13, and blue indicates cell nuclei. The bar is 50 μm. b and c IF was performed to validate the knockdown efficiency of TRIP13 in SW480 and HCT116 cells. Red and green indicate TRIP13, and blue indicates cell nuclei. The bar is 50 μm assay, in which TRIP13 led to increased colony formation TRIP13 on cell migration and invasion. These assays (Fig. 3c). And knockdown of TRIP13 decreased cell via- showed that cells overexpressing TRIP13 have increased bility (Figs. 4b, c). Furthermore, transwell migration and migration and invasion ability compared with negative invasion assays were employed to assess the influence of control cells (Figs. 3d, e), while knockdown TRIP13 Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 5 of 14 Fig. 3 Upregulation of TRIP13 promotes tumorigenic properties in vitro. a Validation of the efficiency of TRIP13 expression. The upper panel is signal intensity; the lower panel is the quantified results. The values indicate the mean ± standard deviation. p < 0.01. b–e The effects of TRIP13 gain of function on in vitro proliferation (b, c), migration (d), and invasion (e). TRIP13 up-regulation significantly increased cell proliferation, migration and invasion abilities. The values indicate the mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001 induced the opposite effect (Figs. 4d, e). In addition, Figure 1A-D). Taken together, these data indicate that similar results with HCT116 and SW480 cells transduced TRIP13 promotes the proliferation, invasion and migra- with LV10-NC and LV10-shTRIP13 (Supplementary tion ability of CRC cells. Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 6 of 14 Fig. 4 Suppression of TRIP13 inhibits the oncogenic phenotype in vitro. a Confirmation of the efficiency of TRIP13 knockdown. The left panel is signal intensity, and the right panel is the quantified results. The values indicate the mean ± standard deviation. p < 0.05. b–e The effects of TRIP13 loss of function on in vitro proliferation (b, c), migration (d), and invasion (e). TRIP13 knockdown inhibits cell proliferation, migration and invasion abilities. The values indicate the mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001 Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 7 of 14 Fig. 5 Altered TRIP13 expression affects tumorigenicity in vivo. a Tumors in nude mice bearing HCT116 cells in different groups. The upper panel is TRIP13 overexpression and the negative control; the lower panel is TRIP13 knockdown and the negative control. Each group contains six mice. b Statistical analysis of tumor volume in different groups. The left panel shows the overexpression and control groups, and the right panel shows the knockdown and control groups. The values indicate the mean ± standard deviation. n = 6/group. **p < 0.01; ***p < 0.001. c Statistical analysis of tumor weight in different groups. The left panel shows the overexpression and control groups, and the right panel shows the knockdown and control groups. The values indicate the mean ± standard deviation. n = 6/group. ***p < 0.001. d Representative staining images of TRIP13 overexpressing tumors. The bars are as indicated. e Representative staining images of TRIP13 knockdown tumors. The bars are as indicated Altered TRIP13 expression affects tumorigenicity in vivo larger tumors than those injected with LV5-NC- The above studies demonstrated that TRIP13 promotes transfected HCT116 cells (Fig. 5a, top panel; Fig. 5b, left the tumorigenic potential of CRC in vitro. Next, we panel). Knockdown of TRIP13 had the opposite effect, explored the effect of TRIP13 on tumorigenesis in vivo and the subcutaneous tumors formed from LV10- using a xenograft model. Nude mice injected with LV5- shTRIP13-infected HCT116 cells were smaller than TRIP13-infected HCT116 cells were found to have much those formed from LV10-NC-infected HCT116 cells Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 8 of 14 (Fig. 5a, bottom panel; Fig. 5b, right panel). These results demonstrated that 14-3-3 proteins play a key role in cell were validated by the tumor weights (Fig. 5c). cycle, apoptosis, and EMT . Collectively, these data Then, H&E staining was used to confirm the tumori- provide strong evidence that TRIP13 plays an important genic characteristics of these subcutaneous tumors part in cell cycle regulation pathways. (Figs. 5d, e, bottom panel), and immunohistochemistry staining was performed with those confirmed cancer tis- TRIP13 modulates EMT in an YWHAZ-regulated way sues. The results showed that TRIP13 was high expressed As YWHAZ interacts with TRIP13 and mediates EMT, in TRIP13 overexpressing tumors and low expressed in TRIP13 promotes EMT, we proposed that TRIP13 may TRIP13 knockdown tumors (Figs. 5d, e, the second panel). mediate EMT dependent on interacting with YWHAZ. In addition, Ki-67 immunohistochemistry staining was To assess this hypothesis, three different siRNAs (si1, si2, used to assess the proliferative index of these tumors. The si3) targeting YWHAZ were applied to transfect TRIP13 results showed that upregulation of TRIP13 increased the overexpressing cells, the known molecular markers of proliferative ability of cells, but downregulation of EMT, TRIP13 and YWHAZ were examined by western TRIP13 suppressed the cell proliferative ability (Figs. 5d, blotting. The results showed that knockdown of YWHAZ e, the first panel). Together, these data suggest that neutralized the up-regulation effect of N-cadherin, β- TRIP13 may promote tumorigenesis in vivo. catenin, snail, and the down-regulation effect of E- cadherin (Fig. 7a). And then down-regulation of TRIP13 is correlated with EMT in CRC cells YWHAZ induced inhibition of migration and invasion EMT is recognized as an important process during ability in both HCT116 and SW480 cells (Fig. 7b,c). carcinoma progression . To determine whether TRIP13 Moreover, the expression of YWHAZ in xenograft tumors has a critical role in the CRC EMT process, we examined was examined by immunohistochemistry, the results changes in known molecular markers of EMT using showed that YWHAZ was high expressed in TRIP13 western blotting in both HCT116 and SW480 cells. The overexpressing tumors and low expressed in TRIP13 results showed that upregulation of TRIP13 increased the knockdown tumors (Figs. 5d, e, the third panel). Fur- expression of N-cadherin, β-catenin and snail but sup- thermore, we evaluated the expression of YWHAZ in the pressed E-cadherin expression (Fig. 6a). In contrast, same 200 CRC FFPE tumor slices, the representative knockdown of TRIP13 suppressed N-cadherin, β-catenin staining scores were shown in Fig. 7d. Spearman corre- and snail and increased E-cadherin expression (Fig. 6b). lation analyses was conducted, and the result showed that Cells transduced with LV10-NC and LV10-shTRIP13 TRIP13 and YWHAZ were positively related (Fig. 7e, r = showed similar results (Supplementary Figure 1E). These 0.532, p < 0.001). These data together indicate that data demonstrated that TRIP13 may promote CRC pro- TRIP13 modulates EMT in an YWHAZ-dependent way. gression by inducing EMT. Discussion TRIP13 interacts with 14-3-3 and modulates cell cycle In the past few decades, a series of biomarkers have To explore the molecular mechanism of TRIP13 in been identified as having prognostic value and therapeutic CRC, we sought to identify potential TRIP13-interacting significance in CRC. However, the overall survival of late proteins. Flag-tagged TRIP13 was expressed in HCT116 stage CRC patients remains poor, and our understanding cells, stained and immunoblotted, and then quantified of the mechanisms of this disease is still inadequate. In using nano-LC-MS/MS (Fig. 6c). A large number of this study, we demonstrated that TRIP13 is a potential proteins were found to interact with TRIP13, and the CRC biomarker. genes encoding these proteins were annotated (Supple- TRIP13, a member of the AAA + ATPase super-family, mentary Excel 1). Then, gene-ontology (GO) Enrichment is located at 5p15.33 and encodes a protein with 432 Analysis was performed to annotate the biological pro- amino acids. Studies have shown that TRIP13 is a key cesses in which these genes are involved (Supplementary regulator of meiotic recombination and the spindle Excel 2). We found that these genes were enriched in cell assembly checkpoint because TRIP13 catalyzes the con- cycle process (Fig. 6d, Supplementary Excel 2). Further- version of C-Mad2 to inactive O-Mad2, with the help of comet more, Kyoto Encyclopedia of Genes and Genomes adapter protein p31 to recruit TRIP13 to HORMA 17,18 (KEGG) pathway analysis validated that these genes par- domain-closure motif complexes . Despite playing a ticipated in cell cycle process (Supplementary Excel 3). In key role in meiotic regulation, overexpression or ampli- addition, STRING database shows a clear interaction fication of TRIP13 has been found in multiple human 19,20 network between TRIP13-interacting genes and other cancers . In our study, TRIP13 was found to be highly DNA replication proteins (Fig. 6e). The TRIP13- expressed in CRC tissues compared with adjacent normal interacting genes YWHAQ and YWHAZ are members tissues, and this result was validated by the analysis of of the 14-3-3 superfamily. A previous study has CRC data sets from Oncomine and TCGA. TRIP13 was Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 9 of 14 Fig. 6 TRIP13 regulates G2-M transition and EMT. a, b Western blotting was performed in HCT116 and SW480 cells to determine the change in EMT markers expression upon gain/loss function of TRIP13. The signal intensity and quantitative analysis are as shown. The values indicate the mean ± standard deviation. *p < 0.05; **p < 0.01; ***p < 0.001. c MS analysis of TRIP13-associated proteins. Cell lysate extract from Flag-tagged TRIP13- expressing or control was subjected to IP, and then, the IP complex was subjected to MS analysis. d GO analysis was performed to determine the biological processes in which TRIP13-interacting genes are involved. e The interaction network of TRIP13-interacting genes from STRING database Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 10 of 14 Fig. 7 TRIP13 mediates EMT by interacting with YWHAZ. a Western blotting of EMT markers, TRIP13 and YWHAZ were performed in TRIP13 overexpressing CRC cells which transfected with siRNAs targeting YWHAZ, to determine whether TRIP13 mediates EMT in a YWHAZ dependent way. b, c The effect of knockdown of YWHAZ in TRIP13 overexpressing CRC cells on migration (b), and invasion (c). d YWHAZ IHC scores of the staining intensity level in representative tumor tissues. The bars are as indicated. e Spearman correlation coefficient plots for TRIP13 and YWHAZ. r = 0.532, 95%CI: (0.421–0.627), p < 0.001 Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 11 of 14 first reported as an oncogene in 2014 in head and neck proteins that interact with TRIP13, and then, GO and cancer, and several subsequent studies revealed that KEGG analysis were applied to explore the biological 5–8 TRIP13 plays an oncogenic role in other neoplasms . function and pathway of the genes encoding these pro- Kurita and his colleagues have demonstrated that TRIP13 teins. The results indicated that TRIP13-interacting genes involves in CRC cell proliferation and invasion and may are involved in G2-M cell cycle transition. Cell cycle has be a potential target for CRC treatment , but they only four sequential phases, G1 (preparation for DNA synth- confirmed the oncogenic role of TRIP13 in vitro, and the esis), S (DNA replication), G2 (preparation for mitosis), mRNA level of TRIP13 was analyzed between normal and and M (mitosis), and each phase is highly controlled and tumor tissues from unpaired patients. Here, we demon- regulated by checkpoints, which aim to maintain genomic strated that TRIP13 promotes HCT116 and SW480 cells integrity by repairing damaged DNA before entering proliferation, migration and invasion, as well as tumor- mitosis. If the damaged DNA cannot be repaired cor- igenicity in vivo. Furthermore, we analyzed TRIP13 with rectly, cell cycle will be arrested . Checkpoints at G1-S clinicopathological characteristics in 200 CRC patients, transition and G2-M transition are the most important and found that TRIP13 was significantly associated with cell cycle checkpoints . However, during cancer cell pTNM stage, CEA and CA19-9. pTNM can be considered evolution, this mechanism is abolished due to mutated as the gold standard for cancer treatment and prognostic checkpoints, and then, damaged DNA can enter mitosis, predictor , CEA and CA19-9 is used to detect relapse and which leads to uncontrolled proliferation and a malignant 22 29 follow response to therapy of CRC . Moreover, multi- phenotype . Our study demonstrated that TRIP13 variate analyses showed that TRIP13 can predict overall interacts with YWHAQ and YWHAZ, which are mem- survival of CRC as well as pTNM and CEA. Therefore, bers of the 14-3-3 protein superfamily. YWHAZ, also TRIP13 may serve as a promising biomarker of CRC. named 14-3-3ζ, plays key roles in cell cycle and EMT , The mechanism by which TRIP13 contributes to recent researches indicated that YWHAZ involves in tumors is still poorly understood. In multiple myeloma, tumor progression and could be a prognostic marker for 30,31 researchers found that TRIP13 induces Mad2 degradation kinds of cancers . So we hypothesis that TRIP13 through the Akt pathway and abrogates spindle check- mediates EMT dependent on interacting with YWHAZ, point . As one of the mitotic checkpoint complexes, Mad2 knockdown of YWHAZ in TRIP13 overexpressing CRC is critical for chromosome segregation . Degradation of cells and western blotting of E-cadherin, N-cadherin, β- Mad2 leads to chromosome mis-segregation during catenin and snail were performed, the results showed that mitosis, which ultimately contributes to cancer develop- the up-regulation effect of N-cadherin, β-catenin, snail, ment and chemotherapy resistance . Zhou and his col- and the down-regulation effect of E-cadherin induced by leagues confirmed that the C-MYC/TRIP13/PUMA axis TRIP13 were rescued by YWHAZ knockdown, further- regulates chronic lymphocytic leukemia . PUMA, a pro- more, we found that TRIP13 and YWHAZ were positively apoptotic protein, was reported to be involved in p53- related. This data together confirmed our hypothesis, mediated apoptosis . However, in this study, researchers TRIP13 may promote CRC progression by interacting found that PUMA mediates cell apoptosis in a TRIP13- with YWHAZ to regulate EMT. dependent manner. As the first research indicated that In sum, TRIP13 promotes CRC cell progression in vivo TRIP13 acts as an oncogene, Banerjee and his colleagues and in vitro and indicates poor CRC patient survival. The utilized mass spectrometry and Co-IP to explore how underlying mechanism involves regulation of G2-M TRIP13 promotes head and neck cancer progression, and transition and EMT through interacting with YWHAZ. they found that TRIP13 promotes non-homologous end Our study suggests that TRIP13 is a potential biomarker joining (NHEJ) and induces chemoresistance . Double- for CRC patients and might further assist in therapeutic strand breaks are the most lethal type of DNA damage, decisions regarding CRC treatment. and they are primarily repaired through homologous recombination (HR) or NHEJ . NHEJ promotes cancer Materials and methods and chromosome instability because it is often inaccurate. Data resource It is well recognized that EMT gives rise to the dis- The analyses of TRIP13 in CRC was performed using semination of tumor cells from primary sites, which Oncomine (www.oncomine.org), which comprised of 7 polarized epithelial tumor cells acquired mesenchymal groups. The number of tissues included was 12 normal and cell phenotype and enhanced migratory capacity and 18 tissues in group 1, 12 normal and 70 tumor tissues in invasiveness . So we examined the effect of TRIP13 on group 2, 10 normal and 5 tumor tissues in group 3, 24 expression of critical EMT markers, E-cadherin, N-cad- normal and 45 tumor tissues in group 4, 32 normal and 32 herin, β-catenin and snail, and found that TRIP13 pro- tumor tissues in group 5, 65 normal and 65 tumor tissues motes EMT process. In order to clarify the underlying in group 6, 38 normal and 50 tumor tissues in group 7. We mechanism, Mass spectrometry was performed to analyze also downloaded level-3 RNAseq expression data of 41 Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 12 of 14 tumor and normal paired COAD from TCGA database, Western blot the TRIP13 HTseq-FPKM in tumor and normal samples Protein was prepared by lysing cells in radio immuno- were abstracted and statistical analyses was used by t-test. precipitation buffer containing phosphatase and protease inhibitors (Beyotime Biotechnology, Nantong, China) and Cell culture quantified using a BCA protein assay kit (Beyotime Bio- The human CRC cell lines HCT116 and SW480 were technology, Nantong, China). In brief, 50 µg of protein purchased from Cell Bank of Chinese Academy of Sci- was resolved in 8–15% Tris-SDS-PAGE gels and trans- ences (Shanghai, China) and cultured in Dulbecco’s ferred to PVDF membranes (Millipore, Billerica, USA). modified Eagle’s medium (DMEM) (Gibco, Carlsbad, The membranes were probed with a primary anti-human USA) supplemented with 10% fetal bovine serum (FBS) TRIP13 rabbit antibody (Proteintech, Chicago, USA) at a (Gibco, Carlsbad, USA) and 1% penicillin/streptomycin dilution of 1:1000 and incubated with HRP-conjugated (Gibco, Carlsbad, USA) in a humidified atmosphere of 5% Affinipure goat anti-rabbit secondary antibody (Pro- CO at 37 °C. teintech, Chicago, USA). Immunoreactive proteins were detected using ECL (Millipore, Billerica, USA) and a Bio- Tissue samples and clinicopathological information rad ChemoDoc MP System. The intensity of lanes was CRC tissues and paired normal tissues were obtained assessed using ImageJ (NIH, USA) from 45 patients who underwent surgical resection in the Department of General Surgery, Shanghai Jiao Tong Uni- Cell transfection and lentivirus infection versity Affiliated Sixth People’s Hospital. The fresh samples We successfully established stable TRIP13- were transported in liquid nitrogen and stored in −80 °C overexpressing HCT116 and SW480 cell lines via infec- until protein extraction. FFPE tumor blocks from 200 CRC tion with LV5-TRIP13 or LV5-NC lentivirus that also patients between January 2010 and January 2012 were encoded GFP. And we constructed two different siRNAs obtained from the Department of Pathology. A complete (si1, si2) and then applied them to transfect HCT116 and follow-up was conducted every 6 months until July 2017. SW480 cells using Lipofectamine 2000. Simultaneously, This study was approved by the Ethics Committee of we cloned the second siRNA sequence (si2) that targeted Shanghai Jiao Tong University Affiliated Sixth People’s TRIP13 into an LV10 vector that also encoded RFP. Hospital, and all patients provided informed consent. YWHAZ was knocked down with siRNAs. All of these constructs were purchased from GenePharma (Shanghai, Immunohistochemistry staining and scoring China), and transduction was performed according to the FFPE tumor blocks were cut into 5-μm-thick sections specific manufacturer’s instructions. and mounted on Premiere microscope slides, and then, the slides were heated at 60 °C for 40 min. Depar- Cell viability and colony formation assay affinization and rehydration was performed as previously Cell viability was assessed using an MTT Cell Pro- described . Antigen retrieval was performed with 10 nM liferation and Cytotoxicity Detection Kit (KeyGEN Bio- citrate antigen retrieval solution (Sangon Biotech, TECH, Nanjing, China). Colony formation ability was Shanghai, China) at 95 °C for 10 min. After non-specific assessed on 6 or 12-well plates for 10–14 days, and interactions were blocked with 3% goat serum, TRIP13 colonies were fixed with methanol and then stained with rabbit polyclonal antibody (Abcam, Cambridge, USA) was crystal violet. used at a dilution of 1:150, YWHAZ rabbit polyclonal antibody (Proteintech, Chicago, USA) was used at a Transwell migration and invasion assay dilution of 1:500, Ki-67 rabbit polyclonal antibody (Pro- Transwell assays were performed with 8-μm poly- teintech, Chicago, USA) with a dilution of 1:400, then carbonate transwell filters (Coring, Cambridge, USA). incubated with the slides overnight at 4 °C. Five random Briefly, 3–5×10 cells were seeded in the upper chamber fields in each slide were selected and evaluated indepen- without FBS, and the lower chamber was filled with 600 µl dently by two pathologists. A semi-quantitative scoring of DMEM containing 10% FBS. After 24–48 h of incu- system was used to evaluate the staining results basing on bation, cells on the lower membrane were set in 4% the percentage and intensity of positively stained cells. paraformaldehyde and stained with crystal violet. Then, 5 The intensity was scored as follows: 0-negative, 1-weak, 2- random fields were selected and photographed. moderate, and 3-strong. The frequency of positively stained cells was defined as: 0-less than 5%, 1–5 to 25%, Immunofluorescence assay 2–26 to 50%, 3–51 to 75%, and 4-greater than 75%. For After the indicated tumor cells crawled on the slide, statistical analysis, scores of 0 to 7 were considered low treatmented with siRNA or lentivirus, the CRC cells were expression, and scores of 8 to 12 were considered high fixed in 4% paraformaldehyde for 15 min. Then, the slides expression. were subjected to 0.2%Triton® X–100 (Sigma Aldrich, Official journal of the Cell Death Differentiation Association Sheng et al. Cell Death and Disease (2018) 9:402 Page 13 of 14 St. Louis, USA) for 20 min and washed with PBST. serum-free DMEM (Gibco, Carlsbad, USA), and a total of Blocking was carried out in 5% bovine serum, and then 5×10 cells were injected subcutaneously into the right samples were incubated with anti-TRIP13 antibody flank of mice. The length and width of the resulting (Proteintech, Chicago, USA) at a dilution of 1:50 over- tumors were measured every 3 days starting 6 days after night at 4 °C. Then, the cells were incubated with Alexa- injection. Tumor volume was calculated using the formula labeled secondary antibodies (anti-rabbit IgG (H + L), F (length × width )/2. After 3 weeks, mice were sacrificed, (ab’)2 fragment (Alexa Fluor® 488 Conjugate)/anti-rabbit and the tumors were collected and weighed. The experi- IgG (H + L), F(ab’)2 fragment (Alexa Fluor® 594 Con- mental procedures were approved by the Ethical Com- jugate) 1:1000, Cell Signaling Technology, Danvers, MA, mittee of Shanghai Jiao Tong University Affiliated Sixth USA) for 30 min at room temperature. Nuclei were People’s Hospital. stained with DAPI (1:5000, Cell Signaling Technology, Danvers, USA). Following three final rinses with PBST, Statistical analysis the cells were imaged with a fluorescence microscope The data are presented as the mean ± standard deviation (Olympus, Japan). of at least three independent experiments. Difference between variables were assessed with a two-tailed t-test or Co-immunoprecipitation and peptide preparation χ analysis. The effect of clinical variables on patient HCT116-Flag-TRIP13 cells were lysed (CST, Danvers, survival was analyzed using Cox proportional hazards USA), and the protein was isolated. TRIP13 complex regression analyses, and Kaplan-Meier survival analysis proteins were purified with anti-Flag M2 magnetic was used to compare CRC patient survival with the log- beads (Sigma-Aldrich, St. Louis, USA). The co- rank test. For correlation, we used Spearman correlation. immunoprecipitation assay procedure was performed p < 0.05 was considered statistically significant. according to the manufacturer’s instructions. Then, the Acknowledgements TRIP13 complex components were eluted in blue loading This work was supported by Shanghai Municipal Education Commission- buffer (CST,Danvers,USA) and SDS-PAGE wasperformed Gaofeng Clinical Medicine Grant Support (no.20172023), the Collaborative Innovation Center for Translational Medicine at Shanghai Jiao Tong University (mini-PROTEAN precast gels, Bio-Rad). The protein lanes School of Medicine (no.TM201519), Shanghai Pujiang Program were visualized with Coomassie brilliant blue staining. (no.16PJ1408200), Natural Science Foundation of Shanghai (no.16ZR1449600), Peptide preparation for mass spectrometric analysis was National Natural Science Foundation of China (no.81602689). performed according to a previously described process . Author details Department of General Surgery, Shanghai Jiao Tong University Affiliated Sixth Mass spectrometry and peptide data analysis People’s Hospital, Shanghai 200233, China. State Key Laboratory of Medical The prepared peptides were analyzed using nano-LC- Genomics, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China MS/MS. LC separations were performed on an Easy nano LC system (Thermo Scientific, Bremen, Germany). Eluted Conflict of interest peptides were directly analyzed using tandem mass The authors declare that they have no conflict of interest. spectrometry (MS/MS) on an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Scientific, Bremen, Ger- Publisher's note many) equipped with a nano-electrospray ion source. Springer Nature remains neutral with regard to jurisdictional claims in Proteome Discoverer 2.0 (Thermo Scientific) software was published maps and institutional affiliations. used to analyze the raw Xcalibur file generated from the Supplementary Information accompanies this paper at https://doi.org/ mass spectrometer. 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