Published OnlineFirst February 2, 2011; DOI: 10.1158/0008-5472.CAN-10-3557 Cancer Tumor and Stem Cell Biology Research Serglycin Is a Theranostic Target in Nasopharyngeal Carcinoma that Promotes Metastasis Xin-Jian Li1, Choon Kiat Ong5, Yun Cao1,2, Yan-Qun Xiang3, Jian-Yong Shao1,2, Aikseng Ooi9, Li-Xia Peng1, Wen-Hua Lu1, Zhongfa Zhang11, David Petillo9, Li Qin4, Ying-Na Bao1, Fang-Jing Zheng1, Claramae Shulyn Chia8, N. Gopalakrishna Iyer6, Tie-Bang Kang1, Yi-Xin Zeng1, Khee Chee Soo7, Jeffrey M. Trent10, Bin Tean Teh5,9, and Chao-Nan Qian1,3,5,10 Abstract Nasopharyngeal carcinoma (NPC) is known for its high-metastatic potential. Here we report the identification of the proteoglycan serglycin as a functionally significant regulator of metastasis in this setting. Comparative genomic expression profiling of NPC cell line clones with high- and low-metastatic potential revealed the serglycin gene (SRGN) as one of the most upregulated genes in highly metastatic cells. RNAi-mediated inhibition of serglycin expression blocked serglycin secretion and the invasive motility of highly metastatic cells, reducing metastatic capacity in vivo. Conversely, serglycin overexpression in poorly metastatic cells increased their motile behavior and metastatic capacity in vivo. Growth rate was not influenced by serglycin in either highly or poorly metastatic cells. Secreted but not bacterial recombinant serglycin promoted motile behavior, suggesting a critical role for glycosylation in serglycin activity. Serglycin inhibition was associated with reduced expression of vimentin but not other epithelial–mesenchymal transition proteins. In clinical specimens, serglycin expression was elevated significantly in liver metastases from NPC relative to primary NPC tumors. We evaluated the prognostic value of serglycin by immunohistochemical staining of tissue microarrays from 263 NPC patients followed by multivariate analyses. High serglycin expression in primary NPC was found to be an unfavorable independent indicator of distant metastasis-free and disease-free survival. Our findings establish that glyco- sylated serglycin regulates NPC metastasis via autocrine and paracrine routes, and that it serves as an independent prognostic indicator of metastasis-free survival and disease-free survival in NPC patients. Cancer Res; 71(8); 1–11. Ó2011 AACR. Introduction lymph nodes (LN) or even distant organs at the time of diagnosis (6). However, the molecular mechanisms underlying Nasopharyngeal carcinoma (NPC) is a common malignancy NPC metastasis are poorly understood. in southern China and Southeast Asia (1, 2). NPC has the Serglycin is a proteoglycan consisting of a core protein to highest metastasis rate among head and neck cancers (3–5), which negatively charged glycoaminoglycan (GAG) chains of with the majority of the patients having metastases to regional either chondroitin sulfate or heparin are attached (7, 8). The core protein containing 158 amino acid residues can be divided into 3 domains: a signal peptide domain, an N-terminal domain Authors' Affiliations: 1State Key Laboratory of Oncology in South China, with unknown function, and a C-terminal domain (9). The Departments of 2Pathology and 3Nasopharyngeal Carcinoma, Sun functions of serglycin in various cells depend on the type and 4 Yat-sen University Cancer Center, Guangzhou, Guangdong; Division of size of the GAG chains decorating the core protein (8, 10–19). Pharmacoproteomics, Institute of Pharmacy and Pharmacology, Univer- sity of South China, Hengyang, Hunan, China; 5NCCS-VARI Translational Serglycin mRNA or protein has been detected in normal Research Program, 6Department of Surgical Oncology, and 7Division of hematopoietic, endothelial, and embryonic stem cells (11, 14, 8 Medical Sciences, National Cancer Center Singapore; Department of 20–23). Serglycin is thought to be important for homeostasis General Surgery, Singapore General Hospital, Singapore; 9Laboratories of Cancer Genetics and 10Genome Biology, Van Andel Research Institute of positively charged components (e.g., proteases) in storage (VARI), Grand Rapids, Michigan; and 11Center for Systems and Computa- granules due to its negatively charged GAG chains (24–28). tional Biology, The Wistar Institute, Philadelphia, Pennsylvania In cytotoxic lymphocytes or natural killer T cells, a macro- Note: Supplementary data for this article are available at Cancer Research molecular complex of granzyme B and perforin complexed Online (http://cancerres.aacrjournals.org/). with serglycin induces the apoptosis of target cells (29–34). Corresponding Author: Chao-Nan Qian, Department of Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, 651 Dongfeng East Serglycin has been associated with tumorigenesis in acute Road, Guangzhou, Guangdong 510060, P.R. China. Phone: 86-20- myeloid leukemia (AML) and myeloma, and it is found to be a 87343457; Fax: 86-20-87343624. E-mail: [email protected] selective marker for distinguishing AML from Philadelphia doi: 10.1158/0008-5472.CAN-10-3557 chromosome-negative chronic myeloproliferative disorders. It Ó2011 American Association for Cancer Research. is also highly expressed by multiple myeloma cell lines (35, 36). www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2011 American Association for Cancer Research. Published OnlineFirst February 2, 2011; DOI: 10.1158/0008-5472.CAN-10-3557 Li et al. Although the involvement of serglycin in tumor metastasis has Histologic evaluation and immunohistochemical been speculated (37), the exact role of serglycin in NPC staining remains unknown. Mouse lymph nodes were routinely fixed and sectioned at 5 The epithelial–mesenchymal transition (EMT), a funda- mm throughout the lymph nodes. To evaluate the microme- mental process in embryonic development, is involved in tastases in mouse lymph nodes, one section in every 20 the metastasis and progression of tumors (38, 39). Activation sequential sections was selected for hematoxylin and eosin of the EMT program, accompanied by an increase in the staining. mesenchymal marker vimentin and loss of the epithelial For immunohistochemical (IHC) staining of serglycin and marker E-cadherin, endows carcinoma cells with enhanced vimentin, paraffin-embedded tissues were sectioned at 5 mm migratory and invasive properties that facilitate dissemination and IHC staining was performed as described previously (42). to permissive niches (39). Briefly, the sections were incubated with a rabbit anti-human serglycin polyclonal antibody (Cat No.: HPA000759, Sigma- Materials and Methods Aldrich; working dilution 1:50) or mouse anti-human E-cad- herin monoclonal antibody (Abcam) overnight at 4C, or Cell culture and cellular growth rate mouse anti-human Vimentin monoclonal antibody (Neomar- Human NPC cell line CNE-2 and its clones (S18, S22, and kers; working dilution 1:100) for 30 minutes at room tem- S26, cultured in less than 50 passages), and SUNE-1 and its perature. An EnVision kit (DAKO) was used to detect the clone 5-8F were maintained in Dulbecco's modified Eagle's primary antibodies followed by 3,3-diaminobenzidine sub- medium supplemented with 10% FBS at 37C. Cellular growth strate visualization and counterstaining with hematoxylin. curves were plotted by using the cellular viability values The intensity of IHC staining in the tumor cells was scored assessed by the MTS method (Cell Titer 96 Aqueous One independently by 2 pathologists by using the semiquantitative Solution Cell Proliferation Assay solution; Promega). IRS (immunoreactive score) scale according to Remmele and Stegner (43), which takes into account both the intensity of the In vitro migration and invasion assays color reaction (no staining ¼ 0; weak staining ¼ 1; moderate Wound healing assays and transwell assays were used to staining ¼ 2; strong staining ¼ 3) and the percentage of evaluate the migration and invasion abilities of the cells (40). stained cells (0% ¼ 0; 1%–10% ¼ 1; 11%–50% ¼ 2; 51%–80% ¼ See the Supplementary Methods for more details. 3; 81%–100% ¼ 4). The average value from the 2 referees was used as the final score. Detection of serglycin in conditioned medium A total of 2 Â 106 cells were plated in 100-mm culture plates Quantitative PCR and incubated for 48 hours in a regular medium, then replaced The methodology of quantitative PCR is described in the with a serum-free medium and incubated for an additional 24 Supplementary Materials and Methods. The sequences of PCR hours. Ten milliliters of conditioned medium was collected primers used for amplification of serglycin and glyceralde- and concentrated to a volume of 200 mL by using Amicon Ultra hyde-3-phosphate dehydrogenase (GAPDH) were as follows: centrifuge filters (10 kDa molecular weight cutoff pore size; GAPDH forward, 50-AAGGTCATCCCTGAGCTGAA-30; GAPDH Millipore). Twenty microliters of the concentrated condi- reverse, 50-TGACAAAGTGGTCGTTGAGG-30; serglycin for- tioned medium was subjected to SDS-PAGE and blotted ward, 50-TATCCT ACGCGGAGAGCCAGGTAC-30; serglycin with anti-human serglycin (Cat no. H00005552-M03, Abnova) reverse, 50-TTCCGTTAGGAAGCCACTCCC AGATC-30. The antibodies. experiments were performed in triplicate. Human tissues and tissue microarray Lentiviral transduction studies All the human tissue samples were obtained from the Cell lines stably expressing either serglycin short hairpin Department of Pathology, Sun Yat-sen University Cancer Cen- RNA (shRNA) or a scrambled nontarget shRNA were establi- ter (SYSUCC), with prior patient consents and