Knockout Cells and Functional Investigation of MAGEC2 in Tumor Cells
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Establishment of MAGEC2-knockout cells and functional investigation of MAGEC2 in tumor cells Jingjing Wang,1 Xiao Song,1 Chengli Guo, Ying Wang and Yanhui Yin Key Laboratory of Medical Immunology, Ministry of Health, Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China Key words Cancer/testis antigen MAGEC2, a member of the type I melanoma-associated anti- Bioinformatics, CRISPR-Cas systems, gene knockout gen family, is expressed in a wide variety of cancer types but not in normal techniques, MAGEC2, proteomics somatic cells. MAGEC2 has long been recognized as a tumor-specific target, how- Correspondence ever, its functions remain largely unknown. In this study, we established Yanhui Yin, Department of Immunology, Peking Univer- MAGEC2-knockout A375 melanoma cell lines using the CRISPR/Cas9 system. sity Health Science Center, No. 38 Xueyuan Road, Beijing Seven clonal cell lines were generated by using four single guide RNAs targeting 100191, China. the coding region of the MAGEC2 gene, which produced indels that abolished Tel: +86-10-82805648; Fax: +86-10-82801436; MAGEC2 protein expression. To identify the differentially expressed protein pro- E-mail: [email protected] files associated with MAGEC2 loss, isobaric tag for relative quantitation-based 1These authors contributed equally to this work. comparative proteomics experiments were carried out on the MAGEC2-knockcout and control A375 cells. Mining of the proteomics data identified a total 224 Funding Information (61.6% upregulated and 38.4% downregulated) proteins to be significantly Natural Science Foundation of Beijing Municipality, (Grant/Award Number: ‘7142087’) National Natural altered in expression level in MAGEC2-knockcout cells. Ingenuity Pathway Analy- Science Foundation of China, (Grant/Award Number: sis indicated that the significantly altered proteins were involved in critical neo- ‘81472645’). plasia-related biological functions such as cell death, proliferation, and movement. Gene ontology analysis identified “apoptosis signaling” as the top- Received May 23, 2016; Revised September 10, 2016; Accepted September 13, 2016 most upregulated pathway associated with MAGEC2 loss. We showed that knockout or knockdown of the MAGEC2 gene sensitized melanoma cells to tumor Cancer Sci 107 (2016) 1888–1897 necrosis factor-a-induced apoptosis. Interestingly, actin-based motility by Rho and RhoA signaling, known to promote cell migration, were also identified as doi: 10.1111/cas.13082 the top downregulated pathways in MAGEC2-knockout A375 cells. In short, our study provides a suitable cell model for exploring the biological functions of MAGEC2 in malignant cells, and sheds light on the molecular pathway by which MAGEC2 promotes tumor development. he type I melanoma-associated antigens (MAGE-I) are a stranded breaks.(12) Recently, we reported that MAGEC2 binds T group of well-characterized members of the cancer-testis directly to the RING domain protein Rbx1, decreases the antigen family, which are aberrantly expressed in a wide vari- degradation of cyclin E, and promotes cell cycle progression ety of tumors but absent from normal adult tissues other than in tumor cells.(13) The common strategies used in all these immunoprivileged germ-line tissues.(1) Although the cellular studies were to knock down endogenous MAGEC2 in cell and physiological functions of MAGE-I proteins in cancer lines by RNAi. A shortcoming of this approach, however, is have not been fully elucidated, recent studies have indicated that neither siRNA nor shRNA is 100% effective at eliminat- that they are associated with hallmarks of aggressive cancers. ing the expression of a target gene product. To comprehen- Cancer/testis antigen MAGEC2, a member of the MAGE-I sively understand the biological functions of MAGEC2 in protein family, is absent in normal adult somatic tissues but tumor cells, establishing cell lines that completely lack the expressed in many types of tumors.(2–6) Clinical studies indi- expression of MAGEC2 are necessary. cate that expression of MAGEC2 is a potent predictor associ- Recently, the bacterial clustered regularly interspaced short ated with sentinel lymph node metastasis in melanoma,(7) and palindromic repeats (CRISPR)-associated endonuclease 9 nuclear MAGEC2 expression in prostate cancer indicates a (CRISPR/Cas9) system has been successfully used as an easy- higher risk for biochemical recurrence after radical prostatec- to-handle, highly specific, and efficient approach for genome tomy.(8) The function of MAGEC2 in tumor cells was first editing in mammalian cells.(14,15) In this study, we applied the reported by Yang et al. in 2007.(9,10) They found that CRISPR/Cas9 system to human melanoma cell line A375 MAGEC2 promotes cell viability in both mast and melanoma which endogenously expresses MAGEC2, and successfully cell lines. Subsequent studies showed that MAGEC2 can form established MAGEC2-knockout A375 clones. To identify the complexes with RING domain protein TRIM28 and promotes differentially expressed protein profiles associated with loss of the degradation of p53 in a proteasome-dependent manner by MAGEC2 expression, isobaric tag for relative quantitation TRIM28.(11) MAGEC2 was also reported to increase phospho- (iTRAQ)(16) based comparative proteomics experiments were rylation of TRIM28 and improve DNA repair after double- carried out on the MAGEC2-knockout and control A375 cells. Cancer Sci | December 2016 | vol. 107 | no. 12 | 1888–1897 © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made. Original Article www.wileyonlinelibrary.com/journal/cas Wang et al. Analysis with bioinformatics and experimental validation MAGEC2 gene were identified by comparing with the wild- showed that MAGEC2 was involved in tumorigenesis and can- type MAGEC2 sequence. cer development. This is the first study to establish MAGEC2- Analysis of off-target cleavage. Potential off-target sites of knockout cells and thoroughly screen the altered proteins the sgRNAs were predicted using the online off-target search- related with MAGEC2 loss, which will open a new window ing tool, Cas-OFFinder (http://www.rgenome.net/cas-offin- for investigating the contributions of cancer/testis antigen der).(17) As all the gRNAs we designed have at least a three- MAGEC2 in tumorigenesis and cancer development. nucleotide mismatch with the other sequence, the regions with a three-nucleotide mismatch with the MAGEC2 gRNA that were most likely to produce off-target mutations were selected Materials and Methods and subjected to PCR and sequence analysis. The off-target Cells and reagents. Human melanoma cell line A375 were locations for each gRNA and primers to amplify these regions purchased from ATCC (Rockville, MA, USA) and maintained in are listed in Table S2. DMEM (ATCC) supplemented with 10% FBS (Invitrogen, Proteomics sample preparation. The harvested cells were Carlsbad, CA, USA), and human melanoma cell line Hs 695T lysed in 25 mM triethylammonium bicarbonate, 8 M urea, 2% cells were purchased from Cobioer Biosciences Company (Nan- Triton X-100, 0.1% SDS, 50 lg/mL DNase I, 50 lg/mL jing, China) and maintained in MEM (Gibco, Gaithersburg, MD, RNase A, and 19 protease inhibitor cocktail (Pierce, Rockford, USA) supplemented with non-essential amino acids, 1 mM IL, USA). Unsolubilized material was removed by centrifuga- sodium pyruvate, and 10% FBS (Invitrogen). MAGEC2-knock- tion at 18 800g for 1 h at 15°C and the supernatants were col- down Hs 695T cells were established by transfecting pGPU6/ lected. Protein concentrations were determined using 2-D Neo-shMAGEC2 or pGPU6/Neo-shNC vectors and selected Quan kit (GE Healthcare, Piscataway, NJ, USA). using 1.5 mg/mL G418 for 2 weeks. Target sequences are as Isobaric tag for relative quantitation labeling. The iTRAQ follows: MAGEC2 shRNA, 50-CAATTGATACCGCAGATGA- labeling was carried out using an iTRAQ Reagent 8-Plex kit 30; and control shRNA, 50-TTCTCCGAACGTGTCACGT-30. (AB Sciex, Foster City, CA, USA) based on the manufac- Recombinant human tumor necrosis factor-a (TNF-a) was pur- turer’s protocol with minor modifications. Cells lysates from chased from PeproTech (Rocky Hill, NJ, USA) and used at three pairs of MAGEC2-WT (WT1, WT2, and WT3) and 20 ng/mL, and cycloheximide (CHX) was from Sigma-Aldrich MAGEC2-Knockout (KO1, KO2, and KO3) A375 cells were (St. Louis, MO, USA) and used at 10 lg/mL. labeled with iTRAQ labeling reagent 113, 114, 115, 116, 117, MAGEC2 single guide RNA expression vector construction. The and 118, respectively. Briefly, 100 lg protein from each of the MAGEC2 guide RNAs (gRNAs) were designed using the six cell lines was reduced with 2 lL of 50 mM tris-(2-carbox- online CRISPR design tool (http://crispr.mit.edu). For effective yethyl) phosphine at 37°C for 1 h, cysteine blocked with 1 lL gene repression, the first 200 bp of the gene-coding region of of 200 mM methyl methanethiosulfonate at 37°C for 30 min, MAGEC2 were selected to be targeted and submitted to the and digested with 3 lg trypsin (Roche) at 37°C for 15 h. The website, which provided a ranked list of all possible guides in peptides were then dried and labeled with isobaric tags, and the query sequence ordered by faithfulness of on-target activity incubated at room temperature for 2 h before being combined. computed as 100% minus a weighted sum of off-target hit- High-pH reversed-phase HPLC fractionation. The combined scores in the target genome. The top six individual gRNAs, iTRAQ-labeled peptides were subjected to high-pH reversed- which had scores >80, were selected and six pairs of comple- phase separation using a Gemini C18 (4.6 9 250 mm, 5-lm mentary oligos containing these MAGEC2 guide sequences particles) column (Phenomenex, Torrance, CA, USA) on a Shi- and BbsI ligation adapters were synthesized (Table S1).