Research Communication The Ubiquitin Ligase TRIM25 Inhibits Hepatocellular Carcinoma Progression by Targeting Metastasis Associated 1 Protein

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1 Research Communication The Ubiquitin Ligase TRIM25 Inhibits Hepatocellular Carcinoma Progression by Targeting Metastasis Associated 1 Protein Hong-liang Zang Sheng-nan Ren Hong Cao Xiao-feng Tian * Department of General Surgery, The China-Japan Union Hospital, Jilin University, Changchun, Jilin, China Abstract Metastasis associated 1 protein (MTA1) is one of the prime facilitators of metastatic progression in all solid tumors including hepatocellular carcinoma (HCC). However, the underlying regulatory mechanism of MTA1 expression in HCC is not clear. In this study, we evaluated MTA1 transcript and protein expression in HCC and normal hepatic cell lines. The results revealed that MTA1 protein expression had a significantly increase in HCC cell line, HuH6, compared with that in normal hepatic cell line, THLE- 2. Determination of protein half-life using cycloheximide (CHX) treatment did not reveal any statistically significant difference in protein turn-over rates between THLE-2 ( h) and HuH6 ( h) cell lines. MTA1 protein level was stabilized in THLE-2 cells after treatment with MG-132 to levels similar to those observed in HuH6 cells. Mass spectrometric analysis of FLAG immunoprecipitates of FLAG-MTA1 transfected THLE-2 cells after MG-132 treated revealed candidate ubiquitin ligases that were interacting with MTA1. RNAi-mediated silencing of each prospective ubiquitin ligase in THLE-2 cells indicated that knockdown of TRIM25 resulted in stabilization of MTA1 protein, indicating TRIM25 as a putative E3 ligase for MTA1. Coimmunoprecipitation of FLAG-tagged MTA1, but not IgG, in MG-132 treated and untreated THLE-2 cells cotransfected with either FLAG-MTA1 or Myc-TRIM25 revealed robust polyubiquitinated MTA1, confirming that the TRIM25 is the ubiquitin ligase for MTA1 degradation. Overexpression of TRIM25 in HuH6 and RNAi mediated silencing of TRIM25 in THLE-2 cells inhibited and increased the cell migration and invasion, respectively. Analysis of The Cancer Genome Atlas data for assessment of TRIM25 transcript level and MTA1 protein expression in 25 HCC patients confirmed an inverse correlation between the expression of TRIM25 and MTA1. Cumulatively, our data reveal a novel mechanism of post-translational to regulate MTA1 expression in normal hepatic cells, which is repressed in HCC. VC 2017 IUBMB Life, 69(10): , 2017 Keywords: metastasis associated 1 protein; MTA1; hepatocellular carcinoma; TRIM25; ubiquitination Abbreviations: HCC, hepatocellular carcinoma; MTA1, metastasis associated 1 protein; CHX, cycloheximide; EMT, epithelial mesenchymal transition; FBS, fetal bovine serum; qrt-pcr, quantitative real time polymerase chain reaction; IP, immunoprecipitation; SEM, standard error of mean. VC 2017 International Union of Biochemistry and Molecular Biology Volume 69, Number 10, October 2017, Pages *Address correspondence to: Dr. Xiao-feng Tian, Department of General Surgery, The China-Japan Union Hospital, Jilin University, No.282, Xinmin Street, Chaoyang District, Changchun, Jilin , China. Tel: Fax: txfdoctor@163.com Received 15 June 2017; Accepted 10 July 2017 DOI /iub.1661 Published online 31 August 2017 in Wiley Online Library (wileyonlinelibrary.com) Introduction As the fifth most common malignancy worldwide, hepatocellular carcinoma (HCC) incidence has shown an increasing trend over the recent years (1 3). The overall prognosis of HCC patients remains poor due to its high rate of metastasis and recurrence (4,5). Therefore, it is important to identify prognostic and/or diagnostic molecular markers that can be implemented in the prevention and early treatment plan of HCC patients. It has been shown that in human HCC cells, estrogen receptor (ER)-a suppressed cell proliferation and invasion through inhibiting metastasis associated 1 protein (MTA1) transcription (6). Metastasis-associated genes (MTAs) are a gene family which was discovered recently. The MTAs have three subtypes MTA1, MTA2, and MTA3. MTA1, the best characterized member of this family, was first isolated by differential complementary IUBMB Life 795

2 IUBMB LIFE DNA screening using the NF rat mammary adenocarcinoma metastatic cell lines (7). MTA1 is a vital component of histone deacetylation complex and nucleosome remodeling and functions as a transcription factor (8,9). Importantly, MTA1 is a central regulator of epithelial mesenchymal transition (9), one of the pre-requisite for metastatic progression. It has been shown that the overexpression of MTA1 leads to poor prognosis in cancer patients (10,11). However, the precisely regulatory mechanism of MTA1 expression in HCC is not known currently. Materials and Methods Clinical Samples, Tissue Processing, and Ethical Considerations Fresh-frozen and paraffin-embedded HCC tissue samples and corresponding adjacent non-tumorous HCC tissue samples were obtained from 25 Chinese patients at the China Japan Union Hospital, Jilin University, between 2010 and Inclusion criteria were that the HCC was confirmed by pathologist and complete clinical and follow-up data availability. None of the included patients underwent preoperative local or systemic treatment. The Institutional Review Board of the China Japan Union Hospital, Jilin University, approved the protocol of the study. RNAlater (Life Technologies, Shanghai, China) was used to immerse the freshly harvested samples before snap freezing within 30 min post-surgery. The HCC tissue and non-tumorous HCC tissue samples were stored in liquid nitrogen. Cell Culture and Treatment HCC cell lines HuH6 and normal hepatic cell line THLE-2 were purchased from the ATCC (Manassas, VA) and cultured in Dulbecco s modified Eagle s media (DMEM) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA) and 100 IU/mL penicillin and 100 lg/ml streptomycin (Gibco). Where indicated, cells were treated with 10 mg/ml cycloheximide (CHX) or 20 mm MG-132 (Sigma-Aldrich, Shanghai, China) for 8 h. RNA Extraction and qrt-pcr Total RNA was isolated from HuH6, THLE-2 cells, HCC tissues, and corresponding adjacent non-tumorous HCC tissue using Trizol reagent. The RevertAid First Strand cdna synthesis Kit (Life Technologies, Shanghai, China) was used to synthesize the first strand. The synthesized cdna was used for quantitative real-time polymerase chain reaction (qrt-pcr) using TaqMan Gene Expression probes (Life Technologies, Shanghai, China) MTA1 (TaqMan Assay ID: Hs _m1) and TRIM25 (TaqMan Assay ID: Hs _m1). Data were normalized to TBP (TaqMan Assay ID: Hs _m1) expression and analyzed by the DDCt method. Gene Construction and Transfection Gene silencing was performed using shrnas from Origene (Rockville, MD). The coding region of TRIM25 and MTA1 cdna obtained from THLE-2 s were sub-cloned into pcmv/myc vector (Addgene, Cambridge, MA) and p3x FLAG-CMV10 vector (Sigma-Aldrich, Louis, MO), respectively. Lipofectamine LTX (Life Technologies, Shanghai, China) was used to transiently transfect cells with indicated plasmids as per the manufacturer s instructions. Cells were harvested after 72 h of transfection and analyzed as indicated. Cell Lysis and Western Blot Cells were lysed using lysis buffer containing 25 mm Tris-HCl ph 7.4, 1 mm EDTA, 150 mm NaCl, 1% NP-40, 5% glycerol, supplemented with complete mini protease inhibitor cocktail (Roche Diagnostics, Indianapolis). Twenty micrograms of whole cell lysate was resolved on a NuPAGE 4 12% gel (Life Technologies, Shanghai, China), and then transferred to an Immobilon poly(vinylidene fluoride) membrane (Millipore, Billerica). The MTA1 antibody (#ab-71153, Abcam, Waltham, MA) and the GAPDH antibody (#ab-9485, Abcam) were used to incubate the blots. The ECL Plus Western blotting substrate and autoradiography film were used to image the blots. Immunoprecipitation THLE-2 cells were transiently transfected with Myc-TRIM25 or Myc-UBE4A and FLAG-MTA1 to detect ubiquitinated-mta1. For immunoprecipitation (IP), whole cell lysates were obtained as above for IP. Cell lysates (1,000 lg) were incubated at 48C overnight with 10 lg mouse anti-flag antibody (Sigma- Aldrich, St. Louis, MO) crosslinked to protein A/G beads (Pierce Crosslink IP Kit, Life Technologies, Grand Island, NY). Total volume was made up to 300 ll with IP lysis buffer. The immune complexes were collected and washed with IP lysis buffer and then eluted with glycine. The eluent were resolved on a 4 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and probed using the anti-flag antibody to detect poly-ubiqutinated MTA1. To determine the E3 ligase that is degrading MTA1 in THLE- 2 cells, MG-132 treated lysates from these cells were immunoprecipitated using anti-flag antibody. MG-132 prevents an ubiquitinated protein from proteasomal degradation, but does not prevent ubiquitination per se. Hence, using MG-132 ensured we had enough amounts of ubiquitinated-mta1 to immunoprecipitate from THLE-2 cells which will also increase the chance of finding the cognate E3 ligase. The immunoprecipitated product was subjected to mass spectrometry using proteomics core services to identify putative E3 ligases interacting with MTA1 protein (data not shown). Mass spectrometry revealed 2192 interacting partners with a false discovery rate, q < 0.05, and 312 interacting partners with a false discovery rate, q < Of the 312 proteins, there were 17 E3 ligases. Prioritization was done using a combination of protein score (cumulative ion score of all identified peptides) and peptide spectrum matches, which resulted in a potential list of 4 E3 ligases. Cell Migration and Cell Invasion Assays The Culturex 96-well cell migration and Culturex 96-well BME cell invasion assay kits were used to detect the ability of cell migration and invasion in vitro, respectively. These experiments were performed according to the manufacturer s recommendations 796 TRIM25 Inhibits Hepatocellular Carcinoma

3 FIG 1 MTA1 is regulated by post-transnational modification in prostate cancer cells. (A) Steady state expression of MTA1 was determined in the HCC cell line HuH6 and normal hepatic cell line THLE-2. Data were normalized to TBP expression. (B) Steady state expression of MTA1 protein in HuH6 and THLE-2 cell lines was determined with Western blot. The GAPDH was used to serve as a loading control. (C) The expression of MTA1 protein in HuH6 and THLE-2 cell lines following treatment with the translation inhibitor CHX for the indicated times showed that the difference in half-life of MTA1 in HuH6 and THLE-2 cells was not significant. GAPDH was used to serve as a loading control. Each panel is representative of at least three independent experiments. (R&D Systems, Shanghai, China). The results were used to analyze the percentage of the cells migration and invasion. The data were expressed as mean 6 standard deviation. The Cancer Genome Atlas Analysis. HCC RNASeqV2 normalized gene expression data on 123 tumor-normal matched pairs were obtained from the The Cancer Genome Atlas (TCGA) data portal (tcga-data.nci.nih.gov/tcga/). R statistical software was used to perform the statistical analysis. Tumornormal log 2 -fold changes were calculated as FC ij 5 log 2 [(T ij 1 1)/(N ij 1 1)], where FC ij represents the fold change for patient i in gene j. T ij and N ij represent the normalized read counts for gene j in tumor and matched normal samples of patient i, respectively. Immunohistochemistry. Tissue specimens from 25 patients with HCC were stained using the anti-mta1 antibody (#ab-71153, Abcam) for the MTA1 expression determination. A pathologist scored the stained slides as percentage staining (0 100%) blinded to the identity of the tissue cores. Statistical Analyses Statistical analyses were performed using SPSS statistics software 20.0 (IBM, Armonk, NY). All data were expressed as mean 6 standard error of mean. Two-sided P < 0.05 were considered statistically significant. Results To study the mechanism of MTA1 expression in HCC, we initially measured the expression of MTA1 mrna and protein in the HCC cell line HuH6 and normal hepatic cell line THLE-2. Even though no significant difference was observed in mrna expression of HuH6 and THLE-2 cells line (THLE-2: /HuH6: ) (Fig. 1A), MTA1 protein expression had a significantly increase in the HuH6 cells compared with that in THLE-2 cells (Fig. 1B). To determine whether the difference in protein expression level between the HuH6 and THLE-2 cells was due to different turn-over rate or stability, the THLE-2 and HuH6 cells were treated with CHX which can inhibit translational elongation. After the treatment of CHX, cell lysates were collected at indicated intervals. As shown in Fig. 1C, no significant difference was found in protein turn-over rate between the two cell lines (MTA1 protein half-life, T 1/ h in THLE-2 and h in HuH6 cells, P > 0.05). Hence, we next determined whether the difference in the level of the MTA1 protein expression between the HuH6 and THLE-2 cells was due to altered degradation kinetics in the two cell lines. Proteasomal degradation inhibitor MG-132 was used to treat the THLE-2 and HuH6 cells. Inhibition of proteasomal degradation increased MTA1 detection in the THLE-2 cells similar to that observed in the HuH6 cells (Fig. 2A). The results suggested that differential targeting by the protein degradation machinery was responsible for altered MTA1 protein expression observed in the HuH6 and THLE-2 cells. To determine whether the E3 ligase can interact with MTA1 in THLE-2 cells, the lysates from THLE-2 cells after treated by MG-132 were immunoprecipitated using anti-mta1 antibody. The immunoprecipitated product was subjected to mass spectrometry to identify putative E3 ligases interacting with MTA1 protein (data not shown). Once the candidate E3 ligases were Zang et al. 797

4 IUBMB LIFE FIG 2 TRIM25 is a putative E3 ligase that causes the degradation MTA1. (A) MTA1 protein expression in HuH6 and THLE-2 cells was determined with Western blot analysis following treatment with the proteasome inhibitor MG-132 for 8 h. GAPDH was used to serve as a loading control. (B) HuH6 cells were transiently transfected with overexpression constructs targeting indicated E3 ligases. Western blot analysis to detect MTA1 expression in mock transfected (control) or transfected with indicated sirnas was performed. GAPDH was used to serve as a loading control. Each panel is a representative of at least three independent experiments. identified, every candidate was overexpressed in the HuH6 cells using expression constructs to detect whether the MTA1 protein degradation can be caused in the HuH6 cells. The results showed that the overexpression of TRIM25 completely silenced MTA1 protein expression in the HuH6 cell line (Fig. 2B), identifying TRIM25 as a putative E3 ligase for MTA1. We next determined whether TRIM25 can ubiquitinate MTA1 protein in vitro in normal hepatic cell. THLE-2 cells were transiently transfected with FLAG-MTA1 and Myc-TRIM25 or Myc-UBE4A (since it was found associated with MTA1 but did not repress MTA1 protein expression in HuH6 cells, Fig. 2B) as indicated in Fig. 3. The cells were treated 6 MG-132 for 6 h after 72 h of transfection. Lysates obtained from these cells were immunoprecipitated with FLAG antibody, resolved by SDS- PAGE, and probed with anti-flag antibody to detect polyubiquitinated MTA1. Only cells treated with MG-132 and cotransfected with both FLAG-MTA1 and Myc-TRIM25 showed potential poly-ubiquitinated smear of MTA1 (Fig. 3A). Probing a similar set of experimental samples with an ubiquitin antibody confirmed that the observed smear was of poly-ubiquitinated MTA1 (Fig. 3B) thus establishing TRIM25 as the E3 ligase for MTA1 protein degradation. We next wanted to determine whether modulating TRIM25 levels in HuH6 cells affect in vitro migration and invasion, which are commonly associated with overexpression of MTA1 in these cells. TRIM25 was overexpressed in HuH6 cells and was silenced by shrna in the THLE-2 cells. We scored each of the individual transfectants for migration (Fig. 4A) and invasion (Fig. 4B) in standard transwell assays. Using these criteria, phase contrast imaging and quantification showed that overexpression of TRIM25 inhibited in vitro migration FIG 3 TRIM25 can ubiquitylate MTA1 in vitro in normal hepatic cell. THLE-2 cells were transiently transfected with FLAG-MTA1 and Myc- TRIM25 or Myc-UBE4A as indicated. Cells were treated 6 MG-132 for 6 h after 72 h transfection. Lysates obtained from these cells were immunoprecipitated with FLAG antibody and resolved by SDS-PAGE. Blots were probed with either anti-flag antibody (A) or anti-ubiquitin antibody (B). The results shown that there were poly-ubiquitinated bands of MTA1 in cells that were transfected with Myc-TRIM25 and treated with MG-132 but were absent where Myc-TRIM25 was not cotransfected or Myc-UBE4A was transfected, or cells were not treated with MG-132. Each panel is a representative of at least three independent experiments. 798 TRIM25 Inhibits Hepatocellular Carcinoma

5 FIG 4 The expression levels of TRIM25 expression regulate in vitro migration and invasion abilities of hepatic cancer cells. THLE-2 cells were transfected with mock or shrna targeting TRIM25 and HuH6 cells were transfected with mock or TRIM25 expression construct. In vitro migration (A) and invasion (B) were determined in the aforementioned cells. A microscope was used to photograph the migrated and invasive cells. The migrated (A) and invasive (B) cells number in every field was counted and represented as percentage of total cells at the beginning of the assay. Error bars, SD. Each panel is representative of at least three independent experiments. ( folds, P ), and invasion (57 6 4, P ) in HuH6 cells. On the other hand, silencing of TRIM25 induced in vitro migration ( folds, P ) and invasion ( folds, P ) in THLE-2 cells. Our results suggested that TRIM25 inhibited in vitro migration and invasion in HCC, which occurred by repression of MTA1 expression. We performed metagenomic analysis of TCGA data to identify the relationship between MTA1 and TRIM25 expression in HCC and verified an inverse correlation between the expression of MTA1 and TRIM25, with the former being higher in HCC patients (Fig. 5A). In fact, when we determined the expression of MTA1 and TRIM25 by immunohistochemistry and qrt-pcr, respectively, in 25 HCC patients, our results indicated a dynamic and inverse correlation between downregulation in the expression levels of TRIM25 and the increase of the MTA1 in HCC tissue specimens (Fig. 5B) (P < 0.05, Pearson correlation r ). Discussion In this study, we found that the protein expression of MTA1 was significantly increased in the HuH6 cells compared with the THLE-2 cells, which was due to differential activity of the E3 ligase TRIM25. TRIM25 can act as the E3 ligase for MTA1 protein degradation. The TRIM25 expression level can regulate the cell migration and invasion in HCC cells. Data mining showed that TRIM25 and MTA1 protein expression follow an inverse correlation in HCC patients. Our findings indicated a novel post-translational mechanism to regulate the expression of MTA1. Studies have shown that MTA1 can function as the facilitator of metastatic progression in all solid tumors. In this study, there was a significant increase in MTA1 protein expression in the HCC cells compared with the normal hepatic cells, which was consistent with other studies. Hamatsu et al. in 2003 first reported the MTA1 expression in HCC patients and they found Zang et al. 799

6 IUBMB LIFE FIG 5 MTA1 and TRIM25 expression are negatively correlated in hepatocellular patients. (A) Metagenomic analysis of the relationship of the expression of MTA1 and TRIM25 in hepatocellular cancer patients. Heat map depicted that the mrna expression of indicated genes between tumor-normal matched pairs for 123 TCGA patients had log 2 -fold change. (B) TRIM25 mrna levels were determined by qrt-pcr in tissue specimens and tumor adjacent normal tissue obtained from 25 patients with HCC. MTA1 expression was determined by immunohistochemistry and scored as percentage staining. TRIM25 expression and MTA1 histo scores were plotted against each other and found to be inversely correlated in these 25 HCC patients. The inverse relationship between TRIM25 and MTA1 in paired samples was demonstrating by Pearson correlation (P < 0.05, Pearson correlation r ). the high expression of the MTA1 gene in tumor tissues of HCC (12). Then Moon et al. revealed that MTA1 was overexpressed in HCC cells versus nonmalignant hepatocytes in 31 of 45 HCC specimens (69%) and they also found that the overexpression of MTA1 was associated with HCC growth and vascular invasion (13). In our study, although there was a significant difference in the MTA1 protein expression between HCC cell line and normal hepatic cell, there was no significant difference in the MTA1 mrna level. These results promoted us to explore the regulation mechanism of MTA1 expression. Then we found that the E3 ligase TRIM25 have the vital role in the regulation of MTA1 expression using the immunoprecipitates experiments and the mass spectrometry method. E3 ligase TRIM25 or tripartite motif containing 25 is a member of TRIM proteins. TRM25 has been found abnormally expressed in many cancers such as ovarian cancer, gastric cancer, and lung cancer (14,15). It has also been shown to interact, but not degrade, and dampen the functional activity of p53 (16). In the lung cancer, the TRIM25 contributes to the lung cancer pathogenesis probably by promoting proliferation and migration of lung cancer cells, and TRIM25 may provide a potential therapeutic intervention for lung cancer (15). In our studies, we determined whether modulating TRIM25 levels in HCC cells affect the cell in vitro migration and invasion. The results showed that the overexpression of TRIM25 in HCC cell lines inhibited in vitro migration and invasion, which was opposite to the results in the lung cancer. Inhibition of cell in vitro migration and invasion after the overexpression of TRIM5 may occur by repression of MTA1 expression. The expression relationship of MTA1 and TRIM25 were also proved in HCC. There was a dynamic and inverse correlation between down-regulation in the expression levels of TRIM25 and the increase of the MTA1 in HCC tissue. In light of these findings, it is imperative to clearly define the pattern of expression and function of TRIM25 in the pathogenesis of HCC. These results indicated that the TRIM25 may act as the diagnostic molecular markers and the target of treatment in the HCC patients. Conclusion In this study, the regulation of the MTA1 expression was explored and found that the TRIM25 as the E3 ligase for MTA1 protein degradation. TRIM25 expression levels could regulate in vitro migration and invasion. Hence, TRIM25 may act as diagnostic molecular marker in the HCC patients. References [1] Siegel, R., Naishadham, D., and Jemal, A. (2013) Cancer statistics, CA Cancer J. Clin. 63, [2] Hanahan, D., and Weinberg, R. A. (2011) Hallmarks of cancer: the next generation. Cell 144, [3] Farazi, P. A., and DePinho, R. A. (2006) Hepatocellular carcinoma pathogenesis: from genes to environment. Nat. Rev. Cancer 6, [4] Aravalli, R. N., Steer, C. J., and Cressman, E. N. 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