(CANCER RESEARCH 57. 4249-4256. October I, 19971 Increased a2,6 Sialylation of N-Glycans in a Transgenic Mouse Model of Hepatocellular Carcinoma' Dominique Pousset, VéroniquePiller,Nicole Bureaud, Michel Monsigny, and Friedrich Nller@ Glycobiologie, Centre de Biophysique Moléculaire,F45071 OrlEans Cedex 02, France ABSTRACT AFP3 levels and in alkaline phosphatase or a rapid deterioration of liver function may be a clue to the presence of the neoplasm (2, 3). Liver cancer is one of the most frequent and lethal malignancies Therefore, the identification of reliable markers for the early diagnosis worldwide. Early detection is hampered by the absence of reliable mark of hepatocellular carcinoma may increase the success rates of surgical era. Mice transgenic for the SV4Olarge T antigen under the control of a liver-specific promoter spontaneously develop well-differentiated hepato resection and other therapies. The search for carbohydrate changes in cellular carcinomas between 8 to 10 weeks of age. They are excellent hepatocellular carcinomas has focused primarily on AFP to distin models to investigate the alterations of protein expression in the early guish the liver-specific glycoprotein from glycoforms that are secreted stages of tumor development and to follow these changes during tumor in other malignancies. It was found that fucosylation of the aspar progressionS In the present study, we analyzed the glycosylation changes agine-linked glucosamine residue of N-glycans was significantly in occurring during tumor development in transgenic mice expressing the creased on AlP from human hepatocellular carcinomas (4, 5). An 5V40 T antigen under the control of the antithrombin ifi promoter. The other fucose-related glycan marker, the Le@epitope, has been reported analysis of serum and liver glycoproteins by an ELISA type assay, using on the cell membranes of poorly differentiated hepatocellular carci the lectin from Sambucusnigra (SNA) as a probe, revealed the presence of nomas in patients expressing high levels of AlP (6, 7). No glycosy increased levels of Neu5Aca2,6Gal@31,4GlcNAc on N-glycans in the hi mor-bearing transgenic mice as compared to controls. On serum glyco lation-related markers have been found for well-differentiated hepa proteins the increase In a2,6 sialylation followed tumor progression, tocellular carcinomas, which generally do not express AFP. reaching up to 10 tImes control levels. However, significantly higher SNA The absence of specific markers makes it almost impossible to binding (2-fold) could already be observed on serum glycoproteins from study the early stages of tumor development in human patients at risk mice exhibiting only microscopically small neoplastic foci. On liver mem for hepatocellular carcinoma. Therefore, the development of animal brane glycoproteins, the increase in a2,6 sialylation was less pronounced, models for the study of tumorigenesis is crucial for our understanding reaching two to three times control values in 6-month-old mice. Western of early events occurring during this process. The first animal models blottingofsenun and liverproteins withradiolabeledSNAshowedthat all were liver cancers induced by chemical carcinogens in the rat (8). glycoproteins that bind the lectin In controls exhibit larger amounts of Since the advent of transgenic techniques, the model of choice has Neu5Aca2,6Gal@31,4GlcNAcon N-glycans in the tumor-bearing mice. This general increase in a2,6 sialylation on all glycoproteins is due to become the mouse expressing a viral or mutated cellular oncogene in the increased activity of the galactosideux2,6 slalyltransferase (ST6GaI I), a strictly tissue-specific manner (9, 10). which specifically transfers Neu5Ac residues in a2,6 linkage to For the study of hepatocellular carcinoma, mice expressing the Galfil,4GIcNAc units on N-glycans. As for the structures synthesized by early region (T/t antigen or Tag) of the SV4O genome under the the enzyme, the increase of ST6GaI I activity in the serum as well as in control of the promoter regions of the albumin, cs-antitrypsin, and liver microsomes of the transgenic mice followed tumor progression. AT!!! genes have been created. All of these mice develop well Interestingly, the activity ofthe galactoslde:a2,3 sialyltransferase (ST3Ga1 differentiated hepatocellular carcinomas (reviewed in Refs. 11 and ifi), whichusesthesameacceptorsubstrate(Gal@1,4GlcNAc),wasun 12). The tumor develops first in microscopically small foci at multiple changed in the earlier stages of tumor development but decreased in the sites throughout the liver. It is characterized by the classical sequence serum and in liver microsomes from later stages. Using a rat ST6GaI I cDNA as a probe, Northern blots of total RNA extracted from the livers ofevents: hyperplasia, dysplasia, adenoma, and carcinoma. In contrast ofcontrol and transgenic mice revealed an increased (4-fold) expression of to other transgenic models of hepatocellular carcinoma, the ATIII the ST6GaJI gene. The single transcripts detected In both normal and SV4O-Tag transgene-induced tumors occasionally develop lung me cancerous liver showed identical size. tastasis and thus show all of the characteristics of an advanced tumor. The proliferating foci develop into nodules that grow until the liver INTRODUCTION reaches about 10 times its normal size. The tumors are composed exclusively of hepatocytes without any elements of the biliary epithe Hepatocellular carcinoma is the most frequent cancer in many parts hum. The tumors are well differentiated, secrete normal amounts of of the world, whereas it is relatively uncommon in Europe or in the albumin, and do not express AFP (1 1). United States. It is rarely cured by surgical resection, and other Recently, a similar transgenic model of rat hepatocellular carci therapies are usually administered as part of clinical trials (1). Success noma has been reported, which shows also all of the characteristics of rate is low, however, due to the absence of other reliable early a well-differentiated tumor. In addition, the comparison of several diagnostics, and the most common primary diagnostic is the detection tumor markers from focal and neoplastic lesions of the livers of rats of an increase in hepatic mass. In about one-half of the patients at risk treated with chemical carcinogens and from transgenic rats revealed (cirrhosis or hepatitis B or C infection), a progressive increase in many similarities between the two experimental neoplasms ( 12). All of these reports clearly show that animals expressing the 5V40- Received 5/19/97; accepted 7/30/97. Tag in a strictly liver-specific manner are excellent models for hep Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage charges. This article must therefore be hereby marked advertisement in accordance with atocellular carcinoma and may, therefore, serve as experimental sys 18 U.S.C. Section 1734 solely to indicate this fact. tems to identify markers of tumorigenesis. Carbohydrate changes on I This work was supported by grants from the Association pour Ia Recherche sur Ic Cancer, Biotechnocentre, the Conseil Regional de la Region Centre, and the Ministèrede l'Enseignement SupéneurCtde Ia Recherche (to F. P.). D. P. is supported by a fellowship 3 The abbreviations used are: AFP, a-fetoprotein; SV4O-Tag, SV4O T antigen; ATIII, from the Association pour la Recherche sur le Cancer. antithrombin III; Neu5Ac. N-acetylneuraminic acid; Gal. galactose; GlcNAc, N-acetyl 2 To whom requests for reprints should be addressed, at Glycobiologie, Centre de glucosamine; ST6Ga1 I, CMP-Neu5Ac:Gal@3l,4GlcNAca2,6 sialyltransferase; ST3GaI Biophysique Moléculaire,rueCharles Sadron, F4507l Orleans Cedex 02, France. Phone: III, CMP-Neu5Ac:Gal@l,3(4)GlcNAca2,3 sialyltransferase: SNA, Sambucus nigra ag (33) 238 25 76 43; Fax: (33) 238 69 00 94; E-mail: [email protected]. glutinin; AGP, a1-acid glycoprotein; PMSF, phenylmethylsulfonyl fluoride. 4249 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1997 American Association for Cancer Research. GLYCOSYLATIONINMURINEHEPATOCELLULARCARCINOMA glycoproteins and glycolipids are associated with tumor development The pellet was freeze-dried and extracted three times with methanol at 70°C (13, 14) and may be used as tumor markers (15, 16). However, very and dried in the vacuum at room temperature. The material was suspended in little is known about glycosylation changes in mouse hepatocellular 5 ml of Pronase buffer [150 mM Tris-HCI (pH 8.0), 75 mM NaCI, and 2 inst carcinoma. Therefore, we screened by lectin-ELISA serum and liver CaC12Iby vortexing and ultrasonication. A 50-pi aliquot of a solution of membrane glycoproteins from normal mice and from mice transgenic Pronase E (10 mg/mI in Pronase buffer) was added, and the mixture was kept at 37°Cfor20 h. After adjusting the pH to 8.0 with 2 si Tris-base, another for the T-antigen from SV4O under the dependence of the human aliquot of Pronase E was added, and the incubation continued for 24 h. The ATIII promoter and analyzed a variety of glycosyltransferases. The addition of Pronase and the incubation were repeated one more time. After specific activity of most glycosyltransferases tested was unchanged in centrifugation, the clear supernatant was heated in a boiling water bath for 5 the tumor-bearing mice, whereas the a2,6 sialyltransferase (ST6Gal I) mm and stored at 4°C.The Pronase digests were applied to a column of was dramatically increased. Because increased sialylation has been Sephadex