Generally, most of ovarian cancer cannot be detected until large scale and remote metastasis occurs, which is the major cause of high mortality in ovarian cancer. Therefore, it is urgent to discover metastasis-related biomarkers for the detection of ovarian cancer in its occult metastasis stage. Altered glycosylation is a universal feature of malignancy and certain types of glycan structures are well-known markers for tumor progressions. Thus, this study aimed to reveal specific changes of N-glycans in the secretome of the metastatic ovarian cancer. We employed a quantitative glycomics approach based on metabolic stable isotope labeling to compare the differential N-glycosylation of secretome between an ovarian cancer cell line SKOV3 and its high metastatic derivative SKOV3-ip. Intriguingly, among total 17 N-glycans identified, the N-glycans with bisecting GlcNAc were all significantly decreased in SKOV3-ip in comparison to SKOV3. This alteration in bisecting GlcNAc glycoforms as well as its corresponding association with ovarian cancer metastatic behavior was further validated at the glycotransferase level with multiple techniques including real-time PCR, western blotting, transwell assay, lectin blotting and immunohistochemistry analysis. This study illustrated metastasis-related N-glycan alterations in ovarian cancer secretome in vitro for the first time, which is a valuable source for biomarker discovery as well. Moreover, N-glycans with bisecting GlcNAc shed light on the detection of ovarian cancer in early peritoneal metastasis stage which may accordingly improve the prognosis of ovarian cancer patients.
References
[1]
Ozols RF, Bookman MA, Connolly DC, Daly MB, Godwin AK, et al. (2004) Focus on epithelial ovarian cancer. Cancer Cell 5: 19–24. doi: 10.1016/s1535-6108(04)00002-9
[2]
Bowtell DD (2010) The genesis and evolution of high-grade serous ovarian cancer. Nat Rev Cancer 10: 803–808. doi: 10.1038/nrc2946
[3]
Siegel R, Naishadham D, Jemal A (2013) Cancer statistics, 2013. CA Cancer J Clin 63: 11–30. doi: 10.3322/caac.21166
[4]
He Y, Wu X, Liu X, Yan G, Xu C (2010) LC-MS/MS analysis of ovarian cancer metastasis-related proteins using a nude mouse model: 14-3-3 zeta as a candidate biomarker. J Proteome Res 9: 6180–6190. doi: 10.1021/pr100822v
[5]
Woodward ER, Sleightholme HV, Considine AM, Williamson S, McHugo JM, et al. (2007) Annual surveillance by CA125 and transvaginal ultrasound for ovarian cancer in both high-risk and population risk women is ineffective. BJOG 114: 1500–1509. doi: 10.1111/j.1471-0528.2007.01499.x
[6]
Ma S, Shen K, Lang J (2003) A risk of malignancy index in preoperative diagnosis of ovarian cancer. Chin Med J (Engl) 116: 396–399.
[7]
Torres JC, Derchain SF, Faundes A, Gontijo RC, Martinez EZ, et al. (2002) Risk-of-malignancy index in preoperative evaluation of clinically restricted ovarian cancer. Sao Paulo Med J 120: 72–76. doi: 10.1590/s1516-31802002000300003
[8]
Doig T, Monaghan H (2006) Sampling the omentum in ovarian neoplasia: when one block is enough. Int J Gynecol Cancer 16: 36–40. doi: 10.1111/j.1525-1438.2006.00273.x
[9]
Kenny HA, Kaur S, Coussens LM, Lengyel E (2008) The initial steps of ovarian cancer cell metastasis are mediated by MMP-2 cleavage of vitronectin and fibronectin. J Clin Invest 118: 1367–1379. doi: 10.1172/jci33775
[10]
Tetsche MS, Dethlefsen C, Pedersen L, Sorensen HT, Norgaard M (2008) The impact of comorbidity and stage on ovarian cancer mortality: a nationwide Danish cohort study. BMC Cancer 8: 31. doi: 10.1186/1471-2407-8-31
[11]
Aletti GD, Gallenberg MM, Cliby WA, Jatoi A, Hartmann LC (2007) Current management strategies for ovarian cancer. Mayo Clin Proc 82: 751–770. doi: 10.4065/82.6.751
[12]
Fuster MM, Esko JD (2005) The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer 5: 526–542. doi: 10.1038/nrc1649
[13]
Hart GW, Copeland RJ (2010) Glycomics hits the big time. Cell 143: 672–676. doi: 10.1016/j.cell.2010.11.008
[14]
Hakomori SI, Cummings RD (2012) Glycosylation effects on cancer development. Glycoconj J 29: 565–566. doi: 10.1007/s10719-012-9448-4
[15]
Ohtsubo K, Marth JD (2006) Glycosylation in cellular mechanisms of health and disease. Cell 126: 855–867. doi: 10.1016/j.cell.2006.08.019
[16]
Granovsky M, Fata J, Pawling J, Muller WJ, Khokha R, et al. (2000) Suppression of tumor growth and metastasis in Mgat5-deficient mice. Nat Med 6: 306–312. doi: 10.1038/73163
[17]
Pochec E, Litynska A, Amoresano A, Casbarra A (2003) Glycosylation profile of integrin alpha 3 beta 1 changes with melanoma progression. Biochim Biophys Acta 1643: 113–123. doi: 10.1016/j.bbamcr.2003.10.004
[18]
Guo HB, Lee I, Kamar M, Pierce M (2003) N-acetylglucosaminyltransferase V expression levels regulate cadherin-associated homotypic cell-cell adhesion and intracellular signaling pathways. J Biol Chem 278: 52412–52424. doi: 10.1074/jbc.m308837200
[19]
Pinho SS, Reis CA, Paredes J, Magalhaes AM, Ferreira AC, et al. (2009) The role of N-acetylglucosaminyltransferase III and V in the post-transcriptional modifications of E-cadherin. Hum Mol Genet 18: 2599–2608. doi: 10.1093/hmg/ddp194
[20]
Handerson T, Camp R, Harigopal M, Rimm D, Pawelek J (2005) Beta1,6-branched oligosaccharides are increased in lymph node metastases and predict poor outcome in breast carcinoma. Clin Cancer Res 11: 2969–2973. doi: 10.1158/1078-0432.ccr-04-2211
[21]
Dall’Olio F, Chiricolo M (2001) Sialyltransferases in cancer. Glycoconj J 18: 841–850. doi: 10.1023/a:1022288022969
[22]
Varki NM, Varki A (2007) Diversity in cell surface sialic acid presentations: implications for biology and disease. Lab Invest 87: 851–857. doi: 10.1038/labinvest.3700656
[23]
Schultz MJ, Swindall AF, Bellis SL (2012) Regulation of the metastatic cell phenotype by sialylated glycans. Cancer Metastasis Rev 31: 501–518. doi: 10.1007/s10555-012-9359-7
[24]
Lin S, Kemmner W, Grigull S, Schlag PM (2002) Cell surface alpha 2,6 sialylation affects adhesion of breast carcinoma cells. Exp Cell Res 276: 101–110. doi: 10.1006/excr.2002.5521
[25]
Seales EC, Jurado GA, Brunson BA, Wakefield JK, Frost AR, et al. (2005) Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility. Cancer Res 65: 4645–4652. doi: 10.1158/0008-5472.can-04-3117
[26]
Christie DR, Shaikh FM, Lucas JAt, Lucas JA 3rd, Bellis SL (2008) ST6Gal-I expression in ovarian cancer cells promotes an invasive phenotype by altering integrin glycosylation and function. J Ovarian Res 1: 3. doi: 10.1186/1757-2215-1-3
[27]
Zhu Y, Srivatana U, Ullah A, Gagneja H, Berenson CS, et al. (2001) Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro. Biochim Biophys Acta 1536: 148–160. doi: 10.1016/s0925-4439(01)00044-8
[28]
Takahashi M, Kuroki Y, Ohtsubo K, Taniguchi N (2009) Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins. Carbohydr Res 344: 1387–1390. doi: 10.1016/j.carres.2009.04.031
[29]
Gu J, Sato Y, Kariya Y, Isaji T, Taniguchi N, et al. (2009) A mutual regulation between cell-cell adhesion and N-glycosylation: implication of the bisecting GlcNAc for biological functions. J Proteome Res 8: 431–435. doi: 10.1021/pr800674g
[30]
Zhao Y, Nakagawa T, Itoh S, Inamori K, Isaji T, et al. (2006) N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrin-mediated cell migration. J Biol Chem 281: 32122–32130. doi: 10.1074/jbc.m607274200
[31]
Isaji T, Gu J, Nishiuchi R, Zhao Y, Takahashi M, et al. (2004) Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration. J Biol Chem 279: 19747–19754. doi: 10.1074/jbc.m311627200
[32]
Song Y, Aglipay JA, Bernstein JD, Goswami S, Stanley P (2010) The bisecting GlcNAc on N-glycans inhibits growth factor signaling and retards mammary tumor progression. Cancer Res 70: 3361–3371. doi: 10.1158/0008-5472.can-09-2719
[33]
Meany DL, Chan DW (2011) Aberrant glycosylation associated with enzymes as cancer biomarkers. Clin Proteomics 8: 7. doi: 10.1186/1559-0275-8-7
[34]
Abbott KL (2010) Glycomic analysis of ovarian cancer: past, present, and future. Cancer Biomark 8: 273–280.
[35]
Gubbels JA, Belisle J, Onda M, Rancourt C, Migneault M, et al. (2006) Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors. Mol Cancer 5: 50.
[36]
Casey RC, Oegema TR Jr, Skubitz KM, Pambuccian SE, Grindle SM, et al. (2003) Cell membrane glycosylation mediates the adhesion, migration, and invasion of ovarian carcinoma cells. Clin Exp Metastasis 20: 143–152.
[37]
Yue T, Goldstein IJ, Hollingsworth MA, Kaul K, Brand RE, et al. (2009) The prevalence and nature of glycan alterations on specific proteins in pancreatic cancer patients revealed using antibody-lectin sandwich arrays. Mol Cell Proteomics 8: 1697–1707. doi: 10.1074/mcp.m900135-mcp200
[38]
Wada Y, Azadi P, Costello CE, Dell A, Dwek RA, et al. (2007) Comparison of the methods for profiling glycoprotein glycans–HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study. Glycobiology 17: 411–422. doi: 10.1093/glycob/cwl086
[39]
Orlando R (2013) Quantitative analysis of glycoprotein glycans. Methods Mol Biol 951: 197–215. doi: 10.1007/978-1-62703-146-2_13
[40]
Mechref Y, Hu Y, Desantos-Garcia JL, Hussein A, Tang H (2013) Quantitative glycomics strategies. Mol Cell Proteomics 12: 874–884. doi: 10.1074/mcp.r112.026310
[41]
Orlando R, Lim JM, Atwood JA 3rd, Angel PM, Fang M, et al. (2009) IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells. J Proteome Res 8: 3816–3823. doi: 10.1021/pr8010028
[42]
Fang M, Lim JM, Wells L (2010) Quantitative Glycomics of Cultured Cells Using Isotopic Detection of Aminosugars with Glutamine (IDAWG). Curr Protoc Chem Biol 2: 55–69. doi: 10.1002/9780470559277.ch090207
[43]
Zhang W, Wang H, Tang H, Yang P (2011) Endoglycosidase-mediated incorporation of 18O into glycans for relative glycan quantitation. Anal Chem 83: 4975–4981. doi: 10.1021/ac200753e
[44]
Qian Y, Zhang X, Zhou L, Yun X, Xie J, et al. (2012) Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Glycoconj J 29: 399–409. doi: 10.1007/s10719-012-9408-z
[45]
Atwood JA 3rd, Cheng L, Alvarez-Manilla G, Warren NL, York WS, et al. (2008) Quantitation by isobaric labeling: applications to glycomics. J Proteome Res 7: 367–374. doi: 10.1021/pr070476i
[46]
Machado E, Kandzia S, Carilho R, Altevogt P, Conradt HS, et al. (2011) N-Glycosylation of total cellular glycoproteins from the human ovarian carcinoma SKOV3 cell line and of recombinantly expressed human erythropoietin. Glycobiology 21: 376–386. doi: 10.1093/glycob/cwq170
[47]
Alley WR Jr, Vasseur JA, Goetz JA, Svoboda M, Mann BF, et al. (2012) N-linked glycan structures and their expressions change in the blood sera of ovarian cancer patients. J Proteome Res 11: 2282–2300. doi: 10.1021/pr201070k
[48]
Ceroni A, Maass K, Geyer H, Geyer R, Dell A, et al. (2008) GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res 7: 1650–1659. doi: 10.1021/pr7008252
[49]
Kameyama A, Kikuchi N, Nakaya S, Ito H, Sato T, et al. (2005) A strategy for identification of oligosaccharide structures using observational multistage mass spectral library. Anal Chem 77: 4719–4725. doi: 10.1021/ac048350h
[50]
Wu W, Sun Z, Wu J, Peng X, Gan H, et al. (2012) Trihydrophobin 1 phosphorylation by c-Src regulates MAPK/ERK signaling and cell migration. PLoS One 7: e29920. doi: 10.1371/journal.pone.0029920
[51]
Liu H, Xu J, Zhou L, Yun X, Chen L, et al. (2011) Hepatitis B virus large surface antigen promotes liver carcinogenesis by activating the Src/PI3K/Akt pathway. Cancer Res 71: 7547–7557. doi: 10.1158/0008-5472.can-11-2260
[52]
Miwa HE, Song Y, Alvarez R, Cummings RD, Stanley P (2012) The bisecting GlcNAc in cell growth control and tumor progression. Glycoconj J 29: 609–618. doi: 10.1007/s10719-012-9373-6
[53]
Yu D, Wolf JK, Scanlon M, Price JE, Hung MC (1993) Enhanced c-erbB-2/neu expression in human ovarian cancer cells correlates with more severe malignancy that can be suppressed by E1A. Cancer Res 53: 891–898.
[54]
Clark HF, Gurney AL, Abaya E, Baker K, Baldwin D, et al. (2003) The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment. Genome Res 13: 2265–2270. doi: 10.1101/gr.1293003
[55]
Boersema PJ, Geiger T, Wisniewski JR, Mann M (2013) Quantification of the N-glycosylated secretome by super-SILAC during breast cancer progression and in human blood samples. Mol Cell Proteomics 12: 158–171. doi: 10.1074/mcp.m112.023614
[56]
Zhang Q, Faca V, Hanash S (2011) Mining the plasma proteome for disease applications across seven logs of protein abundance. J Proteome Res 10: 46–50. doi: 10.1021/pr101052y
[57]
Wu CC, Hsu CW, Chen CD, Yu CJ, Chang KP, et al. (2010) Candidate serological biomarkers for cancer identified from the secretomes of 23 cancer cell lines and the human protein atlas. Mol Cell Proteomics 9: 1100–1117. doi: 10.1074/mcp.m900398-mcp200
[58]
Dowling P, Clynes M (2011) Conditioned media from cell lines: a complementary model to clinical specimens for the discovery of disease-specific biomarkers. Proteomics 11: 794–804. doi: 10.1002/pmic.201000530
[59]
Karagiannis GS, Pavlou MP, Diamandis EP (2010) Cancer secretomics reveal pathophysiological pathways in cancer molecular oncology. Mol Oncol 4: 496–510. doi: 10.1016/j.molonc.2010.09.001
[60]
Makridakis M, Vlahou A (2010) Secretome proteomics for discovery of cancer biomarkers. J Proteomics 73: 2291–2305. doi: 10.1016/j.jprot.2010.07.001
[61]
Pavlou MP, Diamandis EP (2010) The cancer cell secretome: a good source for discovering biomarkers? J Proteomics 73: 1896–1906. doi: 10.1016/j.jprot.2010.04.003
[62]
Hu Y, Desantos-Garcia JL, Mechref Y (2013) Comparative glycomic profiling of isotopically permethylated N-glycans by liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 27: 865–877. doi: 10.1002/rcm.6512
[63]
Dennis JW, Granovsky M, Warren CE (1999) Protein glycosylation in development and disease. Bioessays 21: 412–421. doi: 10.1002/(sici)1521-1878(199905)21:5<412::aid-bies8>3.0.co;2-5
[64]
Guo R, Cheng L, Zhao Y, Zhang J, Liu C, et al. (2013) Glycogenes mediate the invasive properties and chemosensitivity of human hepatocarcinoma cells. Int J Biochem Cell Biol 45: 347–358. doi: 10.1016/j.biocel.2012.10.006
[65]
Yoshimura M, Nishikawa A, Ihara Y, Taniguchi S, Taniguchi N (1995) Suppression of lung metastasis of B16 mouse melanoma by N-acetylglucosaminyltransferase III gene transfection. Proc Natl Acad Sci U S A 92: 8754–8758. doi: 10.1073/pnas.92.19.8754
[66]
Kang X, Wang N, Pei C, Sun L, Sun R, et al. (2012) Glycan-related gene expression signatures in human metastatic hepatocellular carcinoma cells. Exp Ther Med 3: 415–422. doi: 10.3892/etm.2011.430
[67]
Sato Y, Takahashi M, Shibukawa Y, Jain SK, Hamaoka R, et al. (2001) Overexpression of N-acetylglucosaminyltransferase III enhances the epidermal growth factor-induced phosphorylation of ERK in HeLaS3 cells by up-regulation of the internalization rate of the receptors. J Biol Chem 276: 11956–11962. doi: 10.1074/jbc.m008551200
[68]
Yoshimura M, Ihara Y, Ohnishi A, Ijuhin N, Nishiura T, et al. (1996) Bisecting N-acetylglucosamine on K562 cells suppresses natural killer cytotoxicity and promotes spleen colonization. Cancer Res 56: 412–418.
[69]
Abbott KL, Lim JM, Wells L, Benigno BB, McDonald JF, et al. (2010) Identification of candidate biomarkers with cancer-specific glycosylation in the tissue and serum of endometrioid ovarian cancer patients by glycoproteomic analysis. Proteomics 10: 470–481. doi: 10.1002/pmic.200900537
[70]
Wiegand KC, Shah SP, Al-Agha OM, Zhao Y, Tse K, et al. (2010) ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Med 363: 1532–1543. doi: 10.1056/nejmoa1008433
[71]
Gu J, Nishikawa A, Tsuruoka N, Ohno M, Yamaguchi N, et al. (1993) Purification and characterization of UDP-N-acetylglucosamine: alpha-6-D-mannoside beta 1–6N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase V) from a human lung cancer cell line. J Biochem 113: 614–619.
[72]
Schachter H (1986) Biosynthetic controls that determine the branching and microheterogeneity of protein-bound oligosaccharides. Adv Exp Med Biol 205: 53–85. doi: 10.1007/978-1-4684-5209-9_2
[73]
Yoshimura M, Ihara Y, Matsuzawa Y, Taniguchi N (1996) Aberrant glycosylation of E-cadherin enhances cell-cell binding to suppress metastasis. J Biol Chem 271: 13811–13815. doi: 10.1074/jbc.271.23.13811
[74]
Rebbaa A, Yamamoto H, Saito T, Meuillet E, Kim P, et al. (1997) Gene transfection-mediated overexpression of beta1,4-N-acetylglucosamine bisecting oligosaccharides in glioma cell line U373 MG inhibits epidermal growth factor receptor function. J Biol Chem 272: 9275–9279. doi: 10.1074/jbc.272.14.9275
[75]
Gu J, Zhao Y, Isaji T, Shibukawa Y, Ihara H, et al. (2004) Beta1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells. Glycobiology 14: 177–186. doi: 10.1093/glycob/cwh016
[76]
North SJ, Huang HH, Sundaram S, Jang-Lee J, Etienne AT, et al. (2010) Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity. J Biol Chem 285: 5759–5775. doi: 10.1074/jbc.m109.068353
[77]
Dennis JW, Lau KS, Demetriou M, Nabi IR (2009) Adaptive regulation at the cell surface by N-glycosylation. Traffic 10: 1569–1578. doi: 10.1111/j.1600-0854.2009.00981.x
[78]
Wilken JA, Badri T, Cross S, Raji R, Santin AD, et al. (2012) EGFR/HER-targeted therapeutics in ovarian cancer. Future Med Chem 4: 447–469. doi: 10.4155/fmc.12.11
[79]
Sawada K, Mitra AK, Radjabi AR, Bhaskar V, Kistner EO, et al. (2008) Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target. Cancer Res 68: 2329–2339. doi: 10.1158/0008-5472.can-07-5167
[80]
Kannagi R, Goto Y, Fukui F (2004) In search of the carbohydrate structures on CD44 critical for hyaluronic acid binding - Roles of sialylation and sulfation. Trends in Glycoscience and Glycotechnology 16: 211–23. doi: 10.4052/tigg.16.211
