Published OnlineFirst November 13, 2017; DOI: 10.1158/1078-0432.CCR-17-0352 Biology of Human Tumors Clinical Cancer Research The RARS–MAD1L1 Fusion Gene Induces Cancer Stem Cell–like Properties and Therapeutic Resistance in Nasopharyngeal Carcinoma Qian Zhong1, Zhi-Hua Liu1,2, Zhi-Rui Lin1, Ze-Dong Hu3, Li Yuan1,Yan-min Liu1, Ai-Jun Zhou1, Li-Hua Xu4, Li-Juan Hu5, Zi-Feng Wang1, Xin-Yuan Guan1, Jia-Jie Hao6, Vivian W.Y. Lui7, Ling Guo1,8, Hai-Qiang Mai1,8, Ming-Yuan Chen1,8, Fei Han1,8, Yun-Fei Xia1,9, Jennifer R. Grandis10, Xing Zhang1, and Mu-Sheng Zeng1 Abstract Purpose: Nasopharyngeal carcinoma (NPC) is the most com- pression increased cell proliferation, colony formation, and mon head and neck cancer in Southeast Asia. Because local tumorigenicity in vitro, and the silencing of endogenous RARS- recurrence and distant metastasis are still the main causes of NPC MAD1L1 reduced cancer cell growth and colony formation treatment failure, it is urgent to identify new tumor markers and in vitro. In addition, RARS-MAD1L1 increased the side population therapeutic targets for advanced NPC. (SP) ratio and induced chemo- and radioresistance. Furthermore Experimental Design: RNA sequencing (RNA-seq) was RARS-MAD1L1 interacted with AIMP2, which resulted in activa- applied to look for interchromosome translocation in NPC. tion of FUBP1/c-Myc pathway. The silencing of FUBP1 or the PCR, FISH, and immunoprecipitation were used to examine the administration of a c-Myc inhibitor abrogated the cancer stem cell fusion gene expression at RNA, DNA, and protein levels in NPC (CSC)-like characteristics induced by RARS-MAD1L1. The expres- biopsies. MTT assay, colony formation assay, sphere formation sion of c-Myc and ABCG2 was higher in RARS-MAD1L1–positive assay, co-immunoprecipitation, chromatin immunoprecipita- HNC samples than in negative samples. tion assay, and in vivo chemoresistance assay were applied to Conclusions: Our findings indicate that RARS-MAD1L1 might explore the function of RARS-MAD1L1 in NPC. contribute to tumorigenesis, CSC-like properties, and therapeutic Results: We demonstrated that RARS-MAD1L1 was present in resistance, at least in part, through the FUBP1/c-Myc axis, imply- 10.03% (35/349) primary NPC biopsies and 10.7% (9/84) in ing that RARS-MAD1L1 might serve as an attractive target for head and neck cancer (HNC) samples. RARS-MAD1L1 overex- therapeutic intervention for NPC. Clin Cancer Res; 1–15. Ó2017 AACR. Introduction 1 State Key Laboratory of Oncology in South China, Collaborative Innovation Nasopharyngeal carcinoma (NPC) is a common malignant Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, China. 2Innovation Center of Cancer Medicine, National Institute of Biological epithelial tumor of the nasopharynx in Southeast Asia with high Sciences, Beijing, China. 3Department of Pain Treatment, The Fourth People's invasive and metastatic potential. The incidence of NPC is 27.2/ Hospital of Kunming City, Yunnan, China. 4Department of Oncology and Hema- 100,000 among males and 11.3/100,000 among females in a tology, The First Affiliated Hospital of Guangzhou Medical University, Guangz- high-risk area of South China (1, 2). Although NPC is sensitive to 5 hou, China. Peking University People's Hospital and Peking University Institute radiotherapy, the main causes of NPC treatment failure are local of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplan- recurrence and distant metastasis (3, 4). Therefore, the identifi- tation, Beijing, China. 6State Key Laboratory of Molecular Oncology, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and cation of new tumor markers and therapeutic targets for advanced Peking Union Medical College, Beijing, China. 7School of Biomedical Sciences, NPC is urgently needed. Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong Genome instability, as one of the hallmarks of cancer, might SAR. 8Department of Nasopharyngeal Carcinoma, Sun Yat-Sen University account for gene mutations, alterations in chromosome number, 9 Cancer Center, Guangzhou, China. Department of Radiation Oncology, Sun gene amplifications, and chromosome translocations (5). In NPC 10 Yat-Sen University Cancer Center, Guangzhou, China. Department of Otolar- biopsies, loss of heterozygosity in chromosomes 3p, 9p, 9q, 11q, yngology Head and Neck Surgery and Clinical and Translational Science Insti- – tute, University of California at San Francisco, San Francisco, California. 13q, 14q, and 16q (6 8) and gain regions in chromosomes 1q, 3q, 8q, 12, and 20q (6, 9) have been frequently observed. Note: Supplementary data for this article are available at Clinical Cancer Alterations in the chromosome number in NPC have been shown Research Online (http://clincancerres.aacrjournals.org/). to correlate with NPC progression (6). Q. Zhong, Z.-H. Liu, and Z.-R. Lin contributed equally to this article. Fusion genes as a result of chromosomal translocation, inver- Corresponding Authors: Mu-sheng Zeng, Sun Yat-sen University Cancer Center, sion, or deletion serve as therapeutic targets for many types of 651 Dongfeng East Rd., Guangzhou 510060, China. Phone: 8620-8734-3191; cancer (10). The first characterized fusion gene, BCR-ABL, which is E-mail: [email protected]; and Xing Zhang, [email protected] caused by the Philadelphia translocation, is present in most doi: 10.1158/1078-0432.CCR-17-0352 chronic myelogenous leukemia (CML) cases (11), and treatment Ó2017 American Association for Cancer Research. with imatinib to inhibit the BCR-ABL tyrosine kinase has www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst November 13, 2017; DOI: 10.1158/1078-0432.CCR-17-0352 Zhong et al. Antibodies and reagents Translational Relevance Antibodies against the C-terminal region of MAD1L1 (catalog This study demonstrated that a fusion gene, RARS-MAD1L1, no. A300-355A) were purchased from Bethyl Laboratories. The was present in primary nasopharyngeal carcinoma and head following additional antibodies were used: against the N-terminal and neck cancer biopsies and induces cellular proliferation region of RARS (Novus Biologicals, catalog no. PAB28524), and cancer stem cell properties in NPC. The FUBP1/c-Myc axis Flag M2 (Sigma-Aldrich, catalog no. F1804), AIMP2 (Abcam, was activated by RARS-MAD1L1. These data indicate that the catalog no. F1804), b-actin (Proteintech Group Inc., catalog no. development of drugs targeting this axis via combinatorial 66009-1-1g), ABCG2 (Abcam, catalog no. ab3380), Sox2 (Cell therapy might benefit patients carrying this fusion gene. Signaling Technology, catalog no. 3579), c-Myc (Cell Signaling Technology, catalog no. 5605s), Bmi-1 (Cell Signaling Techno- logy, catalog no. 2933), FUBP1 (Abcam, catalog no. 213528), a-tubulin (Cell Signaling Technology, catalog no. 2125s), glycer- revolutionized treatment for CML patients (12). In solid tumors, aldehyde-3-phosphate dehydrogenase (GAPDH; KangChen Bio- – the TMPRSS2-ERG fusion gene serves as a prognostic marker of tech, catalog no. 60004), and Texas Red-X conjugated wheatgerm fi prostate cancer (13). EML4-ALK was identified in non–small cell agglutinin (WGA; Thermo Fisher Scienti c, catalog no. 11262), lung cancer (NSCLC; ref. 14), and crizotinib, which targets EML4- FITC-conjugated anti-rabbit IgG (Invitrogen). The Myc inhibitor ALK, is associated with a better clinical response (15). (10058-F4) and fumitremorgin C (FTC) were purchased from In NPC, several fusion genes, including FGFR3-TACC3 (16), Selleck Chemicals (catalog no. 7153) and Sigma-Aldrich (SKBO), fl UBR5-ZNF423, CTAGE5-PSMA3 and RARS-MAD1L1 (17), have respectively. Cisplatin and 5- uorouracil were obtained from been identified by RNA-Seq. We previously detected the presence Hospira Australia Pty., Ltd. of the FGFR3-TACC3 fusion gene in approximately 2%–4% of NPC, head and neck squamous cell carcinoma, esophageal squa- FISH analysis mous cell carcinoma and lung cancer samples (16). FGFR3- FISH analysis of fresh tumor tissues or paraffin-embedded TACC3 has been shown to induce oncogenic characteristics by tissue specimens was conducted using a previously described activating FGFR kinase activity and is a potential therapeutic target method (19–21). (16, 18). UBR5-ZNF423 was identified in the C666-1 NPC cell line and in 12/144 NPC tissues. A function analysis has suggested that Cell lines and culture conditions UBR5-ZNF423 might play an important role in tumorigenesis in a The NPC cell lines CNE2, HK1, and S26 were cultured in subset of NPC cases. Whether UBR5-ZNF423 represents a thera- RPMI1640 (Invitrogen) supplemented with 5% FBS (Invitrogen). peutic target in NPC needs further investigation (17). Although C666–1, another NPC cell line, was cultured on CellBIND culture Chung and colleagues reported several other fusion transcripts, flasks (Corning Inc.) in RPMI1640 supplemented with 10% FBS. À À including RARS-MAD1L1, in C666-1 cells (17), we provide the In addition, p53 / mouse embryonic fibroblasts (MEF) and first characterization of RARS-MAD1L1 as a recurrent fusion in 293T cells were cultured in DMEM (Invitrogen) supplemented approximately 10% of primary NPC tumors and head and neck with 10% FBS. All cell lines were cultured in a humidified cancer cases. Furthermore, we demonstrated that RARS-MAD1L1 incubator containing 5% CO2 at 37 C. Cell line authentication might contribute to tumorigenesis and therapeutic resistance