MNAT1 Is Overexpressed in Colorectal Cancer and Mediates P53 Ubiquitin-Degradation to Promote Colorectal Cancer Malignance

Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 https://doi.org/10.1186/s13046-018-0956-3

RESEARCH Open Access MNAT1 is overexpressed in colorectal cancer and mediates p53 ubiquitin- degradation to promote colorectal cancer malignance Shan Zhou1,2†, Jinping Lu2†, Yuejin Li1†, Chan Chen2, Yongqiang Cai2, Gongjun Tan2, Zhengke Peng2, Zhenlin Zhang2, Zigang Dong3, Tiebang Kang4 and Faqing Tang1*

Abstract Background: MNAT1 (menage a trois 1, MAT1), a cyclin-dependent kinase-activating kinase (CAK) complex, high expresses in various cancers and is involved in cancer pathogenesis. However, mechanisms underlying its regulation in carcinogenesis are unclear. Methods: The tissue microarray of colorectal cancer (CRC) was used to evaluate MNAT1 expressions in CRC tissues using immunohistochemistry, CRC cell lines were also detected MNAT1 expression using Western-blotting. MNAT1 and shMNAT1 vectors were constructed, and transfected into CRC cells. Cell growths of the transfected cells were observed using MTT and colony formation. The affects of MNAT1 on p53 expression were analyzed using Western- blotting and Real-time PCR. Immunoprecipitation assay was used to analyze the interaction p53 and MNAT1, and Western-blotting was used to test the effects of MNAT1 on p53 downstream molecules. The apoptosis of CRC cells with MNAT1 or shMNAT1 were analyzed using flow cytometry. BABL/c athymic nude mice were used to observe the effect of MNAT1 on CRC cell growth in vivo. Results: MNAT1 was found to be overexpressed in CRC tissues and cells, and MNAT1 expressions in CRC tissue samples were associated with CRC carcinogenesis and poor patient outcomes. MNAT1-knockin increased CRC cell growth and colony formation, and MNAT1-knockdown dramatically decreased cell motility and invasion. MNAT1 physically interacted with p53, MNAT1 also increased the interaction of MDM2 with p53. Strikingly, MNAT1 mediated p53 ubiquitin-degradation. MNAT1 shortened p53 half-life, and ectopic MNAT1 expression decreased p53 protein stability. Moreover, MNAT1 induced RAD51 and reduced p21, cleaved-caspase3, cleaved-PARP and BAX expression. MNAT1 inhibited CRC cell apoptosis. shMANT1 decreased tumor growths in nude mice following p53 increase. Conclusion: MNAT1 binds to p53, mediates p53 ubiquitin-degradation through MDM2, increases cell growth and decreases cell apoptosis, and finally promotes CRC malignance. MNAT1 binding to p53 and mediating p53 ubiquitin-degradation axis represents a novel molecular joint in the p53 pathway. Keywords: MNAT1,Colorectalcancer,p53,Ubiquitin,Tumorigenesis

* Correspondence: [email protected] †Shan Zhou, Jinping Lu and Yuejin Li contributed equally to this work. 1Department of Clinical Laboratory, Hunan Cancer Hospital &The affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 2 of 17

Background associated with ER expression and patient outcome [25]. Colorectal cancer (CRC) is one of the most common ma- In the present study, we found that MNAT1 is highly lignancies worldwide, with approximately 1.2 million new expressed in CRC tissues, its expression was associated cases and 608,700 deaths every year [1]. Various factors with CRC carcinogenesis and poor patient outcomes. Fur- are involved in CRC incidence. CRC development is char- ther experiments showed that MNAT1 increases CRC cell acterized by an ‘adenoma– carcinoma sequence’.Overex- growth in vitro and in vivo, its mechanism is that MNAT1 pression of specific oncogenes or low expression of tumor induces p53 ubiquitin-degradation. suppressor genes in the epithelium results in the formation of a hyperproliferative mucosa, produces a benign ad- Methods enoma, and eventually forms a carcinoma [2–4]. This Reagents and antibodies process is orchestrated by different proteins, such as, Chemical reagents for molecular biology were purchased Wnt, bone morphogenetic protein (BMP) and transform- from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified ing growth factor (TGF)-β, along with p53 [5]. Alterations Eagle medium (DMEM) and other supplements were molecule pathways, such as cell cycle, cell proliferation, obtained from Life Technologies (Rockville, MD). Anti- and apoptosis are involved in CRC onset. These alter- bodies against p53, p21, PARP, cleaved-PARP, RAD51, ations are responsible for colorectal epithelium carcino- caspase3, cleaved-caspase3, BAX, MDM2, and BCL-2 genesis, which evenly confer individual susceptibility to were purchased from Abnova Company (Shanghai, cancers when they are germlines [6–8]. China). Antibodies against MNAT1, HSP70, GAPDH, MNAT1 (menage a trois 1, MAT1) was initially identi- HA and Flag were purchased from Santa Cruz Biotech- fied as the third subunit besides CDK7 and Cyclin H in nology, Inc. (Santa Cruz, CA) and Cell Signal Technol- cyclin-dependent kinase-activating kinase (CAK) com- ogy, Inc. (Beverly, MA). plex [9–12]. MNAT1 functions as an assembly factor and a substrate specificity-determining factor of CAK to Tissue microarray and immunohistochemical staining promote the stability and activation of CAK [13–15]. Human tissue microarrays containing 80 pairs of CRC Moreover, CAK, as the kinase subunit of general tran- tissues and corresponding adjacent non-tumor tissues, scription factor IIH (TFIIH), is involved in transcription and 20 cases of CRC cancers at various stages were pur- [10]. Activated CAK implicates in phosphorylating and chased from Outdo Biotech Company (Shanghai, China). activating CDKs to ensure cell cycle progression [16], One hundred patients enrolled into this study contained phosphorylating retinoblastoma tumor suppressor pro- 57 males and 43 females. The median age of the patients tein (pRb) to mediate cell cycle G1 exit [17–19]. Import- was 46.5 age years (range 35–76), < 50 age year patients antly, CAK can phosphorylates a series of transcription were 34 cases, > 50 age years patients were 66 cases. The factors, including p53, Oct-1, Oct-2, Oct-3, retinoic acid tumor histology and stages were classified according to receptor alpha (RARα), and peroxisome proliferator-acti- the WHO classification and the TNM staging system of vated receptor gamma (PPARγ), thereby regulating gene the UICC, respectively. Patients in T1-T2 stages were 37 transcription [14, 15]. MNAT1 exerts the above func- cases, and T3-T4 patients were 63 cases. Patients in N0 tions through its distinct domains interacting with stage were 39 cases, and N1-N3 patients were 61 cases. downstream molecules. C-terminal domain of MNAT1 Patients in M0 stage were 34 cases, and M1 patients interacts with the CDK7-Cyclin H complex to stimulate were 66 cases. These tissue microarrays (HcolA180su10) CDK7 kinase activity, the coiled-coil domain of MNAT1 were stained with MNAT1 antibody (dilution 1:5000) as interacts with XPD and XPB to anchor CAK to TFIIH described previously [26]. The stained tissue microarrays core, while N-terminal domain RING finger of MAT1 is were evaluated independently by two pathologists who involved in C-terminal domain (CTD) phosphorylation were blinded to the clinical features and clinical out- of RNA Polymerase II (PolII), which is required for gene come. Each case was scored based on the intensity and promoter release and transcription initiation [20]. Intact percentage of cells. At least 10 high-power fields were MNAT1 expression is associated with cell cycle G1 exit, chosen randomly, and > 1000 cells were counted for each whereas intrinsically programmed or RA-induced MNAT1 section. The intensity of MNAT1 staining was scored as degradation leads to cell cycle arrest, transcription inhib- 0 (no signal), 1+ (weak), 2 (moderate), and 3 (marked). ition and cell differentiation [18, 21–23]. In the inhibition Percentage scores were assigned as 0, negative; 1, 1–25%; of RA-induced granulocytic differentiation, an inhibition 2, 26–50%; 3, 51–75%; and 4, 76–100%. The summed (ex- of MNAT1 degradation mediates p21 expression suppres- tension + intensity) was used as the total score. We sion [23]. Suppressed MNAT1 triggers apoptosis [17]. In grouped all samples into the high expression group (total contrast, MNAT1 overexpression is associated with low score ≥ 2) and the low one (total score<2) according to the p21 expression [24]. Recent reports show that MNAT1 is proteinexpression[27]. Immunohistochemical staining for overexpressed in breast cancer, its expression level is MNAT1 was quantified using German semiquantitative Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 3 of 17

scoring system as described previously [28]. Immunoreac- 100 μg/ml phenylmethanesulfonyl fluoride (PMSF), 10 μ tive score (IRS) was determined using the product of the g/ml aprotinin, and 1 mM sodium orthovanadate]. Cell extent score and the staining intensity score. lysates obtained were centrifuged, and protein concentration of the clarified lysates was measured using Easy Cell lines and cell culture II Protein Quantitative Kit (BCA). 40 μg of the super- CRC cell lines, SW480, HT-29, SW620, DLD1, HCT116, natant protein was separated by 10% SDS-PAGE and − − loVo, RK0, HCT116 p53+/+,HCT116p53 / , and HEK293T transferred onto a nitrocellulose membrane. The blot (an embryonic kidney cell line 293 T) were obtained was blocked with 5% non fat milk, incubated with the from American Type Culture Collection (Maryland). indicated antibody, and then incubated with an appro- All the cell lines were grown in DMEM supplemented priate peroxidase conjugated secondary antibody. The with 10% fetal bovine serum (FBS) at 37 °C and in 5% signal was developed using 4-chloro-1-napthol/3,3-o-di- CO2. aminobenzidine, and relative photographic density was quantified by a gel documentation and analysis system. Plasmids and vectors constructing GAPDH or HSP70 was used as an internal control to MNAT1 DNA fragment was generated by polymerase verify basal expression levels and equal protein loading. chain reaction (PCR) and cloned into pSIN-vector contain- The ratio of the specific proteins to GAPDH orHSP70 ing a FLAG, HA or V5 tag sequence. PT53 was generated was calculated. 100 μg of the clarified supernatants were using PCR and cloned into vector containing HA or FLAG. immunoprecipitated using anti-FLAG-agarose or anti- Short hairpin RNAs (sh) target MNAT1,andshMDM2tar- HA-agarose antibody (Sigma Chemical Co.). MNAT1 or gets MDM2. shMNAT1#1andshMNAT1#2 were designed, p53 in the immunoprecipitated complexes was respect- andshMNAT1andshMNAT1#2sequencesareshownin ively determined by Western-blotting with anti-MNAT1 Additional file 1: Table S1. shMDM2 was designed as de- or anti-p53 antibody. scribed previously [29]. They were synthetized by Gene- Pharma (Shanghai, China) and cloned into pLVX, and Apoptosis analysis then pLVX-shMNAT1#1 and pLVX-shMNAT1#1 were Apoptosis analysis was performed as described previ- obtained. HA-tagged ubiquitin was gifted by Dr. Helen ously [32]. Briefly, 1 × 104 cells of shscramble-HCT116, Piwnica-Worms (Washington University, St. Louis). As shMNAT1#1-HCT116, shMNAT1#2-HCT116, pSIN- described previously [14, 30], the vectors containing HEK293T, and pSIN-MNAT1-HEK293T were seeded various PT53 and MNAT1 domains were generated on six-well plates and cultured to reach 70% confluence, using Quick-Change Site-Directed Mutagenesis Kit and were treated with 10 or 80 μg/ml 5-fluorouracil (Stratagene, California). PCR primers used are listed in (5-FU). After 24 h treatment, the cells were collected by Additional file 2: Table S2. All the mutations were veri- 0.02% trypsin without eathylene diamine tetra acetic acid fied by performing sequencing. (EDTA), and stained with annexin V-EGFP (Enhanced Green Fluorescent Protein) and propidium iodide (Key- Gene transfection and stable transfect of cells Gen Biotec) according to the manufacturer’s recommen- Gene transfection and stable cell line establishment were dations, and analyzed by flow cytometry. performed as described previously [31]. Briefly, 1 × 104 of HCT116 and DLD1 cells were transfected with 2 μg DNA of pSIN, pSIN-MNAT1, pLVX-shMNAT#1, pLVX- MTT and colony formation assays shMNAT1#2 or pLVX-shscramble following the manufac- Cell growth was determined by performing MTT ture’s suggested protocol. HEK293T cells were transfected [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 -H-tetrazo- with pSIN or pSIN-MNAT1. The stably transfected cell lium bromide] assays as described previously [33]. Briefly, lines, pSIN-HCT116, MNAT1-HCT116, pSIN-DLD1, pSIN-HCT116, pSIN-MNAT1-HCT116, pSIN-DLD1, MNAT1-DLD1, shscramble-HCT116, shMNAT1#1- pSIN-MNAT1-DLD1, shscramble-HCT116, shMNAT1#1- HCT116, shMNAT1#2-HCT116, shscramble-DLD1, HCT116, shMNAT1#2-HCT116, shscramble-DLD1, shMNAT1#1-DLD1, shMNAT1#2-DLD1, pSIN-HEK293T, shMNAT1#1-DLD1, and shMNAT1#2-DLD1 cells (1 × and pSIN-MNAT1-HEK293T were obtained by selection 103) were seeded in 96-well microplates. The cells were and further confirmed by assessing MNAT1 expression. cultured for the indicated time, followed by incubation with MTT for 4 h. Optical density (OD) was determined Western-blotting and immunoprecipitation at 450 nm using a microplate reader. Measurements were Western-blotting and immunoprecipitation were per- acquired once per day for 5 d. For the colony-formation formed as described previously [31]. Briefly, 1 × 106 cells assay, the cells were plated at a density of 500 cells/well in were lysed with lysis buffer [1 × PBS, 1% Nonidet P-40, six-well plates and were cultured for 12 d. Colonies were 0.5% sodium deoxycholate, 0.1% SDS, and freshly added fixed in methanol, stained with 0.5% gentian violet, and Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 4 of 17

counted [34]. Results are presented as mean ± SD of three carried out as described in the invasion assay with no independent experiments. coating of matrigel.

Real-time PCR Protein half-life detection Real-time PCR was performed as described previously Protein half-life was determined as described previously μ [37]. Briefly, pSIN- and pSIN- MNAT1-HEK293, shscram- [30]. Briefly, 1 g DNase-treated RNA was reverse tran- # scribed using Revert AidTM First-Strand cDNA Synthesis ble- and shMNAT1 1- LoVo cells were treated with 10 Kit (MBI Fermentas, USA) according to the manufac- mg/mL cycloheximide (CHX), and the treated cells were turer’s instructions. Threshold cycle (Ct) value of each collected at indicated time points after CHX treatment for sample was determined using Platinum SYBR Green 0, 20, 40, 60, 90 and 120 min. Protein of the collected cells qPCR SuperMix-UDG with ROX (Invitrogen) in ABI was extracted for performing Western-blotting with 7900HT Real-Time PCR System (Applied Biosystems, anti-p53 or anti-MNAT1 antibody. GAPDH was used as Foster City, CA). Sequences of primers used are shown in an internal control to verify basal level expression and Additional file 3: Table S3. Relative mRNA expression of equal protein loading. The abundance ratio to HSP70 was each target gene was normalized to the expression of the counted, and half-life time of the proteins was calculated. housekeeping gene GAPDH. Relative mRNA level was Ubiquitination assay calculated as two power values of ΔCt (Ct value of GAPDH Ct of target gene). In vivo ubiquitination assay was performed as described previously [37, 38]. Briefly, HEK293T cells were stable transfected with pSIN-MNAT1, LoVo cells were stable Tumor growth assays in vivo transfected with shscramble, shMNAT1#1orshMNAT1#2. In vivo tumor growth assays were performed as de- The stable cell lines were cotransfected with plasmids ex- scribed previously [35]. Briefly, female BABL/c athymic pressing 3Flag-p53 and HA-ubiquitin. The cells were lysed nude mice (age 4 w) were obtained from an animal cen- in lysis buffer. The cell lysates were centrifuged. The super- ter of Guangdong Province (Guangzhou, China). All ani- natants were immunoprecipitated with anti-Flag agarose, mal experiments were performed according to the and the immunocomplexes were immunoblotted using National Institutes of Health Animal Use Guidelines on anti-HA antibody. the Use of Experimental Animals. The nude mice were subcutaneously injected 2 × 106 cells shscramble- # +/+ # Results HCT116, shMNAT1 1-HCT116p53 or shMNAT1 1- MNAT1 is highly expressed in CRC cells and tissues −/− HCT116p53 , 6 mice per group. Tumor sizes of nude To clarify MNAT1 expression in CRC cells, CRC cell mice were measured every 2 or 3 d, and tumor volume lines, SW480, HT-29, SW620, DLD1, RK0, LoVo, and was estimated. After 17 days, the mice were euthanized, HCT116 cells were detected MNAT1 expression using and the tumors were removed and weighed. Western-blotting. Compared with HEK293T, MNAT1 protein levels were mostly elevated in SW480, HT-29, Cell invasion and motility assay SW620, DLD1, RK0, LoVo, and HCT116 (Fig. 1a). Cell invasion and motility were assayed according to HCT116 and DLD1 had a relatively low MNAT1 expres- the methods described previously with minor modifica- sion, and LoVo had a high expression (Fig. 1a), they tions [36]. Cell invasion and motility of shscramble- were used to perform the next experiments. To clarify HCT116, shMNAT1#1-HCT116, shscramble-DLD1, and whether MNAT1 overexpresses in CRC tissues, a tissue shMNAT1#1- DLD1 cells were detected using Boyden microarray containing 80 pairs of CRC, adjacent chamber invasion assay in vitro. Briefly, for invasion assay, non-tumor tissues, and other 20 CRC tissue samples matrigel (25 mg/50 ml, Collaborattiv Biomedical Products, was used to detect MNAT1 expression. The immunohis- Bedford, MA) was added into the chamber to be 8 mm tochemical results showed that MNAT1 was significantly pore size polycarbonate membrane filters. The cells were high in CRC tissues when compared with the matched trypsinized to be suspension cells, and were seeded into adjacent normal tissues (Fig. 1b, c. p = 0.042). The posi- the Boyden chamber (Neuro Probe, cabin John, MD) at tive rates of MNAT1 expression were compared in nor- the upper part at a density of 1.5 × 104 cells/well in 50 μl mal colorectal, primary CRC, and metastatic CRC of serum-free medium, and then incubated for 12 h at 37 ° tissues. The positive rates of MNAT1 were 11.3% in nor- C. The bottom chamber also contained standard medium mal tissues, 55.9% in primary CRC and 56.1% in meta- with 20% FBS. The cells invaded to the lower surface of static CRC tissues, respectively (Table 1). MNAT1 was membrane were fixed with methanol and stained with significantly upregulated in primary CRC (Table 1. p = hematoxylin and eosin. Invaded cell numbers were 0.031) and metastatic CRC tissues (Table 1. p = 0.033), counted under a light microscope. The motility assay was whereas no difference of MICAL2 between primary CRC Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 5 of 17

Fig. 1 MNAT1 expressions in CRC and its association with CRC patient outcomes. A, MNAT1 expressions were detected in SW480, HT-29, SW620, DLD1, RK0, LoVo, HCT116, and HEK329T using Western-blotting. B, MNAT1 expressions in CRC and non-tumor tissues microarray were detected using immunohistochemstry (IHC). a, negative, scored as 0; b, weakly positive, scored as 1; c, moderately positive, scored as 2; d, positive, scored as 3; e, positive, scored as 4; Scale Bar, 100 μm. C, MNAT1 expression levels in CRC tissue samples were quantified using a German semiquantitative scoring system. Relative expressions in CRC tissues and non-tumor tissues were statistically analyzed using Mann-Whitney U test. The dots represent scores. D, The cut-off value of MNAT1 immunoreactive score is 4.6 according to the ROC curve, and the AUC is 0.933. ROC, receiver operating characteristic; AUC, area under the curve. E, The overall survival of CRC patients with high (49) or low (51) MNAT1 expression. *, p <0.05

and metastatic CRC tissues. In addition, MNAT1 level may nearby tissue invasion) (Table 2. p = 0.882), N stage (lymph be used for the CRC diagnosis, and the area under the node metastasis) (Table 2. p = 0.891), nor M stage (distant curve (AUC) was calculated. Cut off of MNAT1 was 4.6 metastasis) (Table 2. p = 0.897). The patients with high (Fig. 1d. AUC = 0.933, p = 0.013). Sensitivity was 87.6%, and MNAT1 displayed shorter overall survival than low specificity was 67.7%. The association of MNAT1 expres- MNAT1 expression (Fig. 1E, p = 0.011). These data sion with CRC stages was analyzed. MNAT1 expression strongly suggest that high MNAT1 has oncogenic po- was not correlated with T stage (original tumor size and tency and is associated with CRC poor outcomes. Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 6 of 17

Table 1 MNAT1 expressions in normal colorectal, primary CRC, DLD1 were infected with pLVX-shMNAT1#1 and pLVX- and metastatic CRC tissues shMNAT1#2. Western-blotting and real-time PCR were MNAT1 performed to evaluate the efficiency of shMNAT1#1, 2. # n – +% P The results showed that the shMNAT1 1, 2 effectively NC 80 71 9 11.3 blocked MNAT1 protein (Fig. 2D-a) and mRNA ex- CRC 59 22 33 55.9 0.031a pression (Fig. 2D-b, c). After MNAT1 was knockdown, HCT116 (Fig. 2E-a, p < 0.05) and DLD1 (Fig. 2E-b, p < MCRC 41 17 23 56.1 0.033b 0.05) growths were decreased when compared with the NC normal colorectal tissue, CRC primary CRC, MCRC metastatic CRC tissues aCRC versus NC; bMCRC versus NC scramble control. Simultaneously, motility and invasion of the transfected cells were also observed, 10 fields were randomly selected and counted the invaded cells Oncogenic properties of MNAT1 in CRC cells per cell well. The results showed that the motility and HCT116 and DLD1 cells with low MNAT1 expressions invasion of MNAT1-knockdown cells were dramatically were used to investigate MNAT1 function in CRC cell decreased when compared with the scramble group growth. We constructed MNAT1 expression vector, (Fig. 2F-a, b, c; Fig.2G-a, b, c. p <0.05). pSIN-MNAT1. HCT116 and DLD1 cells were transfected with pSIN-MNAT1. MNAT1 was detected in the transfected cells using Western-blotting, and the results MNAT1 down-regulates expressions of p53 displayed that MNAT1 was overexpressed in the trans- The above results suggested that MNAT1 is associated fected cells (Fig. 2A). Viability of the transfected cells with CRC cell growth. We next investigated mechanisms was determined by performing MTT assay. MTT data underlying MNAT1-regulated cell growth, and focused showed that the growth kinetics of HCT116 (Fig. 2B-a. on MNAT1 regulating p53 and its molecular mechan- p < 0.05) and DLD1 cells (Fig. 2B-b. p < 0.05) increased ism. CRC cells were transfected with MNAT1 expression when being transfected with MNAT1. Further, cell col- vectors, p53 and PT53 mRNA expressions were de- ony formation of the transfected cells was detected. tected. The results showed that p53 expressions signifi- MNAT1 dramatically increased colony formation of cantly decreased after being transfected with MNAT1 HCT116 (Fig. 2C-a, b, c. p < 0.05) and DLD1 cells (Fig. (Fig. 3A-a, b), but p53 mRNA expression did not change 2C-d, e, f. p < 0.05). Next, shMNAT1#1 and shMNAT1#2 (Fig. 3B). To observe the dose effect of MNAT1 on p53 were designed to target MNAT1, and pLVX-shMNAT1#1 expression, HEK293T cells were exogenously transfected and pLVX-shMNAT1#2 were constructed. HCT116 and HA-p53 and various dose Flag-MNAT1, and then p53 was detected. p53 expression decreased along with Table 2 MNAT1 expressions in CRC samples at various clinical MNAT1-dose increase, displaying a dose-dependent stages manner (Fig.3C-a, b). To further observe the effect of Characteristic Cases MNAT1 expression p MNAT1 silencing on p53 expression, LoVo cells were Low(%) High(%) transfected with shMNAT1#1 and #2, and p53 expres- All patients sion was detected. The results showed that p53 expres- Gender sion was increased when MNAT1 was silenced (Fig. 3D). Doxorubicin (DOX), an anticancer regent, has been Male 57 35(61.4) 22(38.6) 0.817 proved to increase p53 expression [39]. Dox was Female 43 29(67.4) 14(32.6) used to treat HCT116 cells at various concentrations, Age (yrs) and p53 and MNAT1 expressions were analyzed. < 50 34 16(47.1) 18(52.9) 0.770 MNAT1 gradually decreased along with DOX con- ≥ 50 66 35(53.0) 31(47.0) centration increase, while p53 gradually increased, T stage displaying significantly concentration-dependent (Fig. 3E). In the next, HCT116 cells were transfected with T1-T2 37 19(51.3) 18(48.7) 0.882 pSIN-MNAT1, and then the transfected cells were T3-T4 63 34(53.9) 29(46.1) treated with MG132, a specific proteasome inhibitor N stage to inhibit MNAT1 expression, and p53 expression N0 39 17(43.6) 22(56.4) 0.891 was observed. MG132 substantially rescued the raise N1-N3 61 29(47.5) 32(52.5) of p53 protein level caused by pSIN-MNAT1 (Fig. M stage 3F), further this results were confirmed with DOX reducing MANT1 expression (Fig. 3G). These sug- M0 34 18(52.9) 16(47.1) 0.897 gest that MNAT1 decreases p53 expression by the M1 66 35(53.0) 31(47.0) proteasome. Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 7 of 17

Fig. 2 (See legend on next page.) Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 8 of 17

(See figure on previous page.) Fig. 2 MNAT1 increases CRC cell growth and MNAT1-knockdown decreases cell growth. HCT116 and DLD1 cells were transfected with pSIN-MNAT1 respectively. A, MNAT1 expressions in the transfected cells were detected using Western-blotting. B, Viability of MNAT1 transfected-HCT116 (a) and DLD1 (b) cells was measured using MTT. C, Colony formation of the transfected- HCT116 and DLD1 cells was detected using colony formation assay. a, HCT116 with pSIN; b, HCT116 with pSIN-MNAT1; c, Cell colony numbers of the transfected HCT116; d, DLD1 with pSIN; e, DLD1 with pSIN-MNAT1; f, Cell colony numbers of the transfected DLD1. D, HCT116 and DLD1 cells were transfected with pLVX-shMNAT1#1or pLVX-shMNAT1#2, respectively. MNAT1 protein (a) and mRNA (b, c) in the transfected cells were detected using Western-blotting or Real-time PCR, respectively. E, Viability of shMNAT1 transfected-HCT116 (a) and DLD1 (b) cells was measured using MTT. F, Motility and invasion of HCT116 cells with shMNAT1s were detected with Boyden chamber invasion assay (a), the motility (b) and invasion (c) cells were counted. G, Motility and invasion of DLD1 cells with shMNAT1s were detected (a), the motility (b) and invasion (c) cells were counted. All experiments were repeated three times. Data are presented as means ±S.D. of three independent experiments and were statistically analyzed using Student’sttest.Scale bar, 100 μm. *, p <0.05

MNAT1 interacts with p53 p53 was detectable in MNAT1-immunoprecipitated com- In this step, we first performed endogenous immunopre- plexes, and MNAT1 was also detectable in p53 immuno- cipitation assay to examine whether MNAT1 directly inter- complexes (Fig. 4A). To further confirm the interaction of acted with p53. LoVo cells with high expression MNAT1 MNAT1 with p53, HEK293T cells were contransfected were used to immunoprecipitate p53 and MNAT1, and with HA-p53 and Flag-MNAT1, and then their interaction then p53 and MNAT1 were detected in the immunocom- was determined using immunoprecipitation. Immunopre- plexes using Western-blotting. The results showed that cipitation results showed that MNAT1 bound to p53 (Fig.

Fig. 3 MNAT1 decreases p53 expression. A, HCT116 cells were transfected with Flag-MNAT1 (a), and DLD1 cells were transfected with pSIN-MNAT1 (b), p53 expressions the tranfected cells were detected using Western-blotting. B, PT53 mRNA expressions in the transfected cells were detected using Real-time PCR. C, HEK293T cells were cotransfected with HA-p53 and Flag-MNAT1 at various concentrations, Flag and HA expressions were detected (a). Abundances of Flag and HA were analyzed (b). D, LoVo cells were transfected with shsramble, shMNAT1#1 and #2, and then p53 and MNAT1 expressions in the transfected cells were detected using Western-blotting. E. LoVo cells were treated with COX at the indicated concentrations, and then MNAT1 and p53 expressions were detected. F, HEK293T cells were transfected with pSIN-MNAT1 or blank vector, and then treated with DOX. MNAT1 and p53 expressions were detected using Western-blotting. G, HEK293T cells transfected with pSIN-MNAT1 were treated with DOX and/or MG132, and then MNAT1 and p53 were detected. DOX, Doxorubicin; *, p <0.05 Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 9 of 17

Fig. 4 MNAT1 interacts with p53. A, the lysates of LoVo cells were immunoprecipitated using anti-IgG, anti-MNAT1 or anti-p53 antibodies, and immunoprecipitation products were analyzed by Western-blotting with the indicated antibody. B, HEK293T cells were cotransfected with Flag- MNAT1 and HA-p53. Flag or HA was immunoprecipitated in the transfected cells, and the immunocomplexes were detected using Western- blotting with HA- or Flag-antibody. C, The various construct plasmids of TP53 were constructed using PCR (a). HEK293T cells were cotransfected with Flag-MNAT1 and the indicated plasmids of TP53. Immunoprecipitation was performed using anti-HA antibody, and the immunoprecipitated products were detected using Western-blotting with anti-Flag antibody. D, The plasmids containing various MNAT1 gene segments were constructed (a). HEK293T cells were cotransfected with HA-TP53 and the indicated plasmids of Flag-MNAT1. Immunoprecipitation was performed using anti-Flag antibody, and the immunoprecipitated products were detected using Western-blotting with anti-HA antibody. FL, full length p53; IP, immunoprecipitation; WCL, whole cell lysate

4B). These findings indicated that MNAT1 interacts with containing various MNAT1 domains were constructed p53. Next, we determined the domains of p53 involved in (Fig. 4D-a) and the plasmids were transfected into this interaction using p53- and MNAT1-expressing plas- HEK293T cells, the immunoprecipitation was per- mids. The plasmids expressing various p53 domains were formed to identify the interacting domains of MNAT1. constructed (Fig. 4C-a). HEK293T cells were transfected The results showed that only full-long MNAT1 inter- with the indicated plasmids, and the immunoprecipitation acted with p53, and MNAT1 mutants containing do- was performed to identify the interacting domains of p53. main deletion could not interact with p53 (Fig. 4D-b). The results showed that the domains containing residue 1– 300 displayed a binding band with MNAT1, while the dele- MNAT1 promotes ubiquitin-degradation of p53 tion of residues 100–300 had no binding signal (Fig. 4C-b). The above results suggest that MNAT1 decreases p53 This indicated that the domain of p53 residues 100–300 protein level, while p53 mRNA level did not change, was necessary and sufficient for interaction with MNAT1. indicating that MNAT1 may regulate p53 at post- Simultaneously, to determine the domains of MNAT1 par- transcriptional level. We speculated that MNAT1 af- ticipated in the binding of MNAT1 to p53, the plasmids fects p53 proteolysis. To confirm this, HEK293T cells Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 10 of 17

were co-transfected with v5-MNAT1 and Flag-p53, and and the cotransfection with shMNAT1#1 and shMNAT1#2 then p53-ubiquitin was detected. The results showed that had less p53 ubiquitin-degradation than the single one Flag-p53 poly-ubiquitin was stronger in the MNAT1 trans- (Fig. 5e). The rescue experiments of MNAT1-mediated fect than the blank control (Fig. 5a). Further, we used p53 ubiquitin-degradation were performed. MANT1- shMNAT1 to knockdown MNAT1 expression, and ob- HEK293 cell was treated with MG132, and then p53 ubi- served whether the reduced-MNAT1 decreases p53 quitination was detected. The results showed that p53 ubiquitination. The results showed that Flag-p53 ubi- ubiquitin-degradation significantly increased in the quitination was dramatically decreased when being MNAT1- transfected cell, this p53 ubiquitin-degradation transfected shMNAT1 (Fig. 5b). Additionally, we in- was decreased by MG132 treatment (Fig. 5e). Collectively, vestigated whether MNAT1 could exactly mediate en- these results indicate that MNAT1 increases p53 ubiquiti- dogenous p53 ubiquitination. pSIN-MNAT1 was nation, thus promoting its proteasomal degradation. transfected into HCT116, and p53 ubiquitination was observed. The endogenous p53 ubiquitination signifi- MNAT1 shortens half-time p53 cantly increased when transfected with pSIN-MNAT1 The above findings showed that MNAT1 promotes p53 (Fig. 5c). We also observed the effect of MANT1- ubiquitin-degradation. Moreover ubiquitin-proteasome knockdown on endogenous p53 ubiquitination. p53 is a highly effective protein-degradation pathway in ubiquitin-degradation dramatically decreased (Fig. 5d), eukaryotic cells [40–42]. Next step, we detected the

Fig. 5 MNAT1 promotes p53 ubiquitin-mediated degradation. A, HEK293T cells were cotransfected with HA-Ub, Flag-p53, and v5-MNAT1. Flag was immunoprecipitated in the transfected cells, and p53 was detected using Western-blotting (a), and p53 life-time was calculated (b). B, LoVo cells were cotransfected with HA-Ub, Flag-p53 and pLVX-shMNAT1#1 or pLVX-shMNAT1#2, respectively. The transfected cells were immunoprecipitated with Flag antibody, and p53 in the immunocomplexes were detected. C, HCT116 cells were cotransfected with pLVX-shMNAT1#1 or pLVX-shMNAT1#2, respectively. The transfected cells were immunoprecipitated with p53 antibody, and then ubiquitin in the immunocomplexes was detected. D, HEK293 cells were transfected with pSIN-MNAT1, and p53 in the transfected cells was immunoprecipitated. Ubiquitin in the immunocomplexes was detected. E, HCT116 cells were cotransfected with pLVX-shMNAT1#1and#2. p53 in the transfeccted cells was immunoprecipitated, and its ubiquitination was detected. IP, immunoprecipitation; Ub, ubiquitin; WCL, whole cell lysate Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 11 of 17

half-life time of p53 when MNAT1 knockin and Whether 5-FU-reduced MNAT1 regulates p53 down- MNAT1 knockout. HEK293 cells were transfected with stream molecules was investigated. Fas, a p53 target gene, pSIN-MNAT1, and treated with CHX. The half-life was decreased in the transfect with MANT1, but did not time of endogenous p53 protein was measured in the change in the transfect with shMNAT1 (Fig. 7D). Simul- treated cells. The half-life time of endogenous p53 was taneously, apoptosis in MNAT1-knockdown cells was also shorterinthetransfectwithpSIN-MNAT1thaninthe detected, there was no difference (data not shown). So, control cells (Fig. 6A-a, b). Simultaneously, LoVo cells 5-FU was used to treat MNAT1-knockdown cells, and cell were knockdown MNAT1 with shMANT1#1, and p53 apoptosis was detected using flow cytometry. The results half-life was analyzed. The data showed that p53 showed that shMNAT1#1 and shMNAT1#2 significantly half-life time was longer in the cells with shMNAT1 enhanced 5-FU-induced apoptosis (Fig. 7E-b, c, f. p < transfection than the control (Fig. 6B-a, b). E3 ubiquitin 0.05), and pSIN-MNAT1 reduced 5-FU-induced apoptosis ligase murine double minute (MDM2) is the most crit- (Fig. 7E-e, f. p < 0.05). Collectively, MNAT1 decreases p53 ical negative regulator for p53 protein stability. MDM2 expression, and regulates p53 downstream molecules, re- binds to p53 and ubiquitinates it for proteasomal deg- duced cell apoptosis. radation [43–45]. Therefore, we tested the effect of MNAT1ontheinteractionofMDM2withp53.MDM2 MNAT1 promotes CRC growth in vivo binding to p53 was dramatically increased in the cells The above-mentioned results showed that MNAT1 in- with MNAT1 transfection, while MDM2 interacted creased CRC cell growth and colony formation, and also with p53 was significantly decreased when MNAT1 found MNAT1-mediated p53 ubiquitin-degradation, and knockdown (Fig. 6C-a, b. p < 0.05). To further confirm regulated p53 downstream molecules. In the next step, whether MNAT1-mediated p53 decrease is through we further confirmed whether MNAT1 exerts oncogenic MDM2, stable cell line, MANT1-HEK293 cells were effect in vivo. Shscramble-HCT116, shMNAT1#1- knockdown MDM2 with shMDM2 to observe p53 ex- HCT116 cells were subcutaneously injected into the dor- pression. The results showed that MNAT1 significantly sal flanks of mice. The tumors of mice were measured decreased p53 expression, but p53 decrease was not per 2 d. After 17 days, the mice were euthanized, and significant when MDM2 knockdown (Fig. 6D). These tumor weights were measured. Data showed that the tu- data suggested MNAT1 mediates p53 ubiquitin through mors of mice injected with shMNAT1#1-HCT116 were increasing interaction of MDM2 with p53. smaller than that of the shscramble mice (Fig. 8A, B. p < 0.05). MNAT1 and p53 expression was detected in these MNAT1 regulates p53 downstream genes tumor tissues using immunohistochemistry, and positive The above findings showed that MNAT1 decreases p53 cells were counted in 10 fields of the IHC stained sec- through ubiquitin-degradation. In the next step, we inves- tion under microscopy. Immunohistochemical results tigated whether MNAT1-decreased p53 affects p53 down- showed that MNAT1 expression was low, and p53 was stream genes. HEK293T cells were transfected with high in the tumor tissues of shMNAT1#1-HCT116 when pSIN-MNAT1, and then p53, p21, caspase, PARP, RAD51, compared with the shscramble group (Fig. 8C-a,b. p < BAX, and Bcl2 were tested. p53, p21, cleaved- caspase 3, 0.05). These results indicated that MNAT1 promoted cleaved-PARP and BAX were decreased in the transfected CRC growth through down-regulating p53 in vivo. Sum- cells, and RAD51 was increased (Fig. 7A). To further marily, MNAT1 binds to p53, and mediates p53 probe whether MNAT1 regulating p21, PAPR, BAX, and ubiquitin-degradation through MDM2, decreased p53 RAD51 is through p53, HCT116 p53+/+ and HCT116 functions, and finally promotes CRC growth (Fig. 8D). − − p53 / cells were used to determine whether p53 gene is essential. The results showed that MNAT1-knockdown Discussion increased p53, p21, and BAX expressions, and decreased The adenoma–carcinoma multistage theory has been doc- PARK and RAD51 expression in HCT116 p53+/+, did not umented for CRC carcinogenesis. In this carcinogenesis − − in HCT116 p53 / cells (Fig. 7B). DOX is an anticancer process, mutation activating multiple oncogenes and in- regent, it has proved to inhibit synthesis of RNA and activating tumor-suppressor genes accumulate in normal DNA [46]. The previous data showed that DOX inhibits colonic epithelial cells and cause adenomas [4]. Our re- MNAT1 expression. To further clarify whether DOX sults suggest that MNAT1 is a novel gene in CRC patho- decreased-MNAT1 also regulates p53 downstream mol- genesis. This is based upon the following three results. (1) ecule, HCT116 cells were transfected with shMNAT1 or MNAT1 was highly expressed in CRC cells, and its ex- pSIN-MNAT1, and then treated with DOX. The results pression was associated with advanced CRC development showed that the cleaved PARP was not increased when and low 5-year survival rate. (2) MNAT1 increased CRC transfected with MNAT1, but cleaved PARP was increased cell malignant activity. (3) In vivo, MNAT1-knockdown when being transfected with shMANT1 (Fig. 7C). decreases tumor growth. These results suggested that Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 12 of 17

Fig. 6 MNAT1 shortens half-life time of p53. A, HEK293 cells were transfected with pSIN or pSIN-MNAT1, and then treated with 20 mg/ml CHX for the indicated time points. pSIN served as a control. MANT1 and p53 expressions were detected using Western-blotting (a). Quantitative analysis was conducted on p53 level in Western-blotting, the half-life time of p53 was calculated (b). B, LoVo cells were transfected with pLVX-shMNAT1#1, p53 and MNAT1 expressions in the transfected cells were detected (a). Quantitative analysis was conducted on p53 level, and the half-life time was also calculated (b). C, HEK293 cells were transfected with pSIN-MNAT1, and LoVo cells were transfected with pLVX-shMNAT1#1 or pLVX-shMNAT1#2. p53 was immunoprecipitated, and MDM2 was detected in the immunocomplexes and WCL using Western-blotting (a). Quantitative analysis was conducted on p53 and MNAT1 expressions (b). D, MANT1-HEK293 cells were transfected with shMDM2, and p53, MDM2, MNAT1 expressions were detected in the transfected cells. CHX, cycloheximide; IP, immunoprecipitation; MGM2, murine double minute 2. WCL, whole cell lysate. *, p <0.05

MNAT1 promotes CRC development. Determination function is to promote cell-cycle arrest and induce apop- of the underlying mechanism indicated that MNAT1 tosis when necessary [47]. p53, which is known as the promotes CRC development through downregulating guardian of the genome [48], plays a critical role in indu- p53. cing apoptosis and preventing oncogenesis [49]. p53 is Tumor suppressor p53, encoded by TP53 gene, is a frequently dysregulated in CRC tissues. Moreover, p53 is central player in cellular DNA damage responses and is associated with CRC pathogenesis and advanced TNM mutated in 50 to 55% of human cancers, whose primary stage, lymph nodes metastasis, and low 5-year survival Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 13 of 17

Fig. 7 (See legend on next page.) Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 14 of 17

(See figure on previous page.) Fig. 7 MNAT1 regulates p53 down-stream molecules. A, HEK293T cells were transfected with pSIN or pSIN-MANT1, and then MNAT1, p53, p21, cleaved caspase3, caspase3, cleaved PARP, PARP, BAX, RAD51, and Bcl2 were detected with Western-blotting. pSIN served as a control. B, HCT116 p53+/+ and HCT116 p53−/− cells were transfected with pLVX-shscramble, pLVX-shMNAT1#1orpLVX-shMNAT1#2, respectively. pLVX-shscramble served as a control. MANT1, p53, p21, BAX, PARP, and RAD51 were detected. C, LoVo cells were transfected with pLVX-shMNAT1#1, and HEK293T cells were transfected with pSIN-MANT1. And then the transfected cells were treated with DOX. The treated cells were evaluated MNAT1, PARP, p53 expression. GAPDH served as a loading control. D, LoVo cells were infected with pLVX-shMNAT1#1 or pLVX-shMNAT1#2, and HEK293T cells were transfected with pSIN-MANT1, and then treated with 5-Fu. The treated cells were evaluated MNAT1, p53, and Fas expression. E, The apoptosis of the treated cells was analyzed using flow cytometry, and apoptosis rates were conducted. a, shscramble plus 5-FU; b, shMNAT1#1 plus 5-FU; c, shMNAT1#2plus5-FU;d, blank vector plus 5-FU; e, pSIN-MNAT1 plus 5-FU; f, apoptosis rates of the above treated cell. DOX, Doxorubicin; 5-Fu, 5-fluorouracil; *, p <0.05

Fig. 8 MNAT1 exerts tumor-promoting effects through p53 in vivo. A, shscramble-HCT116 and shMNAT1#1-HCT116 cells were subcutaneously injected into the dorsal flanks of mice, and shscramble-HCT116 served as a control. After 17 days, the mice were euthanized. Representative images are shown (a). After euthanization, the tumors of mice were removed. The removed tumors were weighed (b). B, The tumor growth of shscramble-HCT116 and shMNAT1#1-HCT116 cells in vivo was calculated by tumor volume. C, MNAT1 and p53 expressions in the implanted tumors were detected using immunohistochemistry (IHC) (a), positive cells were counted in 10 fields of the IHC stained section under microscope (b). *, p < 0.05. D, Schematic illustration of MNAT-promoted CRC tumorigenesis. MNAT1 binds to p53, and mediates p53ubiquitin-degaradation, and increases cell growth and decreases cell apoptosis, finally promotes CRC malignance Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 15 of 17

rate [50, 51]. Of the well-known functions of p53, the reduced p53 decreases CRC cell apoptosis and in- mostly highlighted ones are the regulation of cell cycle creases CRC cell growth both in vitro and in vivo, checkpoints and inducing apoptosis under cellular stress thus promoting CRC malignance (Fig. 8D). MNAT1 [52]. Loss of p53 often induces oncogenesis [53–55], and binding to p53 and mediating p53 ubiquitin- promotes tumor initiation and progression [56–58]. In degradation axis represents a novel molecular joint the present study, we found that MNAT1 shortened p53 in the p53 pathway. half-lift, suggesting that MNAT1 may promote CRC carcinogenesis through decreasing p53 function. Determin- Additional files ation of the underlying mechanism indicated that (1) MNAT1 decreases apoptosis through reducing p53 and Additional file 1: Table S1. MNAT1 shRNA sequence. (DOCX 15 kb) that (2) MNAT1 regulated p53 downstream molecules, Additional file 2: Table S2. PCR primers of plasmids construct. (DOCX 16 kb) including p21, PARK, RAD51, and FAS. p21 is critical Additional file 3: Table S3. Primers of Real-time PCR. (DOCX 15 kb) for p53-mediated G1/S boundary cell cycle arrest [59]. p53 mediates cell apoptosis by activating mitochondrial Abbreviations and death receptor-induced apoptotic pathways. The 5-FU: 5-fluorouracil; AUC: Area under the curve; BMP: Bone morphogenetic protein; CAK: Kinase-activating kinase; CHX: Cycloheximide; CRC: Colorectal mitochondrial pathway is mainly regulated by binding to cancer; DOX: Doxorubicin; FBS: Fetal bovine serum; IRS: Immunoreactive Bcl-2, and releasing the cell death factors, BAX and BAK, score; MDM2: Murine double minute; MNAT1: Menage a trois 1; MTT: 3-(4,5- and activate apoptosis [60, 61]. In our study, MNAT1 not dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; OD: Optical density; sh: Short hairpin RNAs only decreased BAX, but also mediated FAS increase. FAS is a component transcriptionally regulated by p53 in the Acknowledgments extrinsic apoptotic pathway FAS [62, 63]. MANT1-medi- We thank various members in Professor Tiebang Kang’s Laboratory for contributions and helpful discussion. We appreciate the contributions and ated apoptosis inhibition may be through the extrinsic helpful discussion of various members in Clinical Laboratory of Zhuhai apoptotic pathway. MNAT1-mediated p21 decrease im- Hospital, Jinan University. plies that MANT1 may arrest cell cycle through p21 to Funding participate in CRC development. These remain to be fur- This work was supported in part by the National Natural Science Foundation ther investigated. of China (81372282, 81872226, 81402368, 81402265,81502346), Program for Interestingly, MNAT1 interacted with p53 and pro- New Century Excellent Talents in University, NCET (NCET-06-0685), Guangdong moted p53 ubiquitination and degradation. MNAT1-me- Natural Science Foundation (S2013010013360). diated p53 degradation may be critical for CRC Availability of data and materials initiation and progression. p53 activity can be regulated The datasets used and/or analysed during the current study are available through ubiquitination, oxidation, phosphorylation, from the corresponding author on reasonable request. – acetylation and methylation [64 66]. The key to the Authors’ contributions regulation of p53 activity is the regulation of its stability, SZ, JL, and YL conducted the study design. YC carried out the assays and which is mainly orchestrated through a network of ubi- collected the samples. SZ and JL performed the statistical analysis. ZG and KT participated the coordination of research and worked as technical consultants. quitination reactions [64, 65] . Among numerous pro- SZ and YL drafted the manuscript. FT and TK revised the manuscript. All authors teins involved in p53 regulation, MDM2 is the major reviewed and approved the final manuscript. negative regulator of p53 level and activity [43, 67]. Ethics approval and consent to participate MDM2 physically interacts with p53 and represses All procedures were consistent with the National Institutes of Health Guide p53-mediated transcriptional activation [44, 45] and in- and approved by institutional board with patients’ written consent. This duce p53 ubiquitination. The E3 ubiquitin ligase MDM2 study was evaluated and approved by the Ethics Committee of the Affiliated Cancer Hospital of Xiangya Medical School, Central South University. All is the most important regulator ubiquitin-mediated deg- animal experiments were performed according to the National Institutes of radation of p53 [67]. MDM2 binds to p53 and ubiquiti- Health Animal Use Guidelines on the Use of Experimental Animals. nates it proteasomal degradation [43–45]. In the present MNAT1 Consent for publication study, knock-in increased MDM2 interaction Informed consent was obtained from all individual participants included in with p53, and this interaction was decreased when the study. MNAT1 knockdown. MDM2-knockdown decreased MNAT1-reduced p53. These data suggest that MNAT1- Competing interests The authors declare that they have no competing interests. mediated p53 ubiquitin-degradation is through increasing the interaction of MDM2 with p53. MDM2 may be a Publisher’sNote critical factor in MNAT1 mediated-p53 ubiquitination. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Conclusion Author details MNAT1 binds to p53, promotes p53 ubiquitin- 1Department of Clinical Laboratory, Hunan Cancer Hospital &The affiliated degradation, and decreases its function. MNAT1- Cancer Hospital of Xiangya School of Medicine, Central South University, Zhou et al. Journal of Experimental & Clinical Cancer Research (2018) 37:284 Page 16 of 17

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