Alterations in Glycopeptides Associated with Herceptin Treatment of Human Breast Carcinoma MCF-7 and T-Lymphoblastoid Cells

The therapeutic humanized monoclonal antibody IgG1 known as Herceptin® has shown remarkable antitumor effects. Although this type of therapy has increased the cancer-free survival of patients, not all tumors respond to this treatment and cancers often develop resistance to the antibody. Despite the fact that Herceptin function has been extensively studied, the precise mechanism underlying its antitumor activity still remains incompletely defined. We previously demonstrated on human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells that monoclonal antibody in combination with Lipoplex consisting of Lipofectamine mixed with plasmid DNA showed a more profound effect on cancer cell viability than antibody alone. The analyses of N-glycans isolated from cancer cells showed dramatic differences in profiles when cells were exposed to Herceptin. Moreover, the investigation of glycosylated peptides from the same cancer cell models after treatment revealed further alterations in the post-translational modifications. Tandem mass spectra obtained from the samples treated confirmed the presence of a series of glycopeptides bearing characteristic oligosaccharides as described in IgG1. However some of them differed by mass differences that corresponded to peptide backbones not described previously and more of them were detected from Herceptin treated samples than from cells transfected with Heceptin/Lipoplex. The results indicate that the presence of Lipoplex prevents antibody transformation and elongates its proper function. The better understanding of the multipart changes described in the glycoconjugates could provide new insights into the mechanism by which antibody induces regression in cancers. The therapeutic humanized monoclonal antibody IgG1 known as Herceptin® has shown remarkable antitumor effects. Although this type of therapy has increased the cancer-free survival of patients, not all tumors respond to this treatment and cancers often develop resistance to the antibody. Despite the fact that Herceptin function has been extensively studied, the precise mechanism underlying its antitumor activity still remains incompletely defined. We previously demonstrated on human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells that monoclonal antibody in combination with Lipoplex consisting of Lipofectamine mixed with plasmid DNA showed a more profound effect on cancer cell viability than antibody alone. The analyses of N-glycans isolated from cancer cells showed dramatic differences in profiles when cells were exposed to Herceptin. Moreover, the investigation of glycosylated peptides from the same cancer cell models after treatment revealed further alterations in the post-translational modifications. Tandem mass spectra obtained from the samples treated confirmed the presence of a series of glycopeptides bearing characteristic oligosaccharides as described in IgG1. However some of them differed by mass differences that corresponded to peptide backbones not described previously and more of them were detected from Herceptin treated samples than from cells transfected with Heceptin/Lipoplex. The results indicate that the presence of Lipoplex prevents antibody transformation and elongates its proper function. The better understanding of the multipart changes described in the glycoconjugates could provide new insights into the mechanism by which antibody induces regression in cancers. Glycosylation of proteins is a ubiquitous type of post-translational modification in living systems. Variations in oligosaccharide structures are associated with many normal and pathological events such as cellular growth, host-pathogen interaction, differentiation, migration, cell trafficking, or tumor invasion (1Bertozzi C.R. Kiessling L.L. Chemical glycobiology.Science. 2001; 291: 2357-2364Crossref PubMed Scopus (1684) Google Scholar, 2Ogata S. Ho I. Chen A. Dubois D. Maklansky J. Singhal A. Hakomori S. Itzkowitz S.H. Tumor-associated sialylated antigens are constitutively expressed in normal human colonic mucosa.Cancer Res. 1995; 55: 1869-1874PubMed Google Scholar). Targeted glycosylation research has become important in the area of developing novel therapeutic approaches (3Danishefsky S.J. Allen J.R. From the laboratory to the clinic: a retrospective on fully synthetic carbohydrate-based anticancer vaccines frequently used abbreviations are listed in the appendix.Angew. Chem. Int. Ed. Engl. 2000; 39: 836-863Crossref PubMed Google Scholar, 4Parodi A.J. Protein glucosylation and its role in protein folding.Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (531) Google Scholar, 5Walsh G. Jefferis R. Post-translational modifications in the context of therapeutic proteins.Nat. Biotechnol. 2006; 24: 1241-1252Crossref PubMed Scopus (706) Google Scholar). The structures of asparagine-linked oligosaccharides in the conserved CH2 region of the constant Fc domain of human immunoglobulin-γ (IgG1) have been shown to affect the pharmacokinetics, antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (6Kanda Y. Yamada T. Mori K. Okazaki A. Inoue M. Kitajima-Miyama K. Kuni-Kamochi R. Nakano R. Yano K. Kakita S. Shitara K. Satoh M. Comparison of biological activity among nonfucosylated therapeutic igg1 antibodies with three different n-linked fc oligosaccharides: the high-mannose, hybrid, and complex types.Glycobiology. 2007; 17: 104-118Crossref PubMed Scopus (293) Google Scholar, 7Suzuki E. Niwa R. Saji S. Muta M. Hirose M. Iida S. Shiotsu Y. Satoh M. Shitara K. Kondo M. Toi M. A nonfucosylated anti-her2 antibody augments antibody-dependent cellular cytotoxicity in breast cancer patients.Clin. Cancer Res. 2007; 13: 1875-1882Crossref PubMed Scopus (92) Google Scholar). In the last decade, many recombinant antibody molecules have been licensed for the treatment of a variety of cancers and chronic diseases (8Jefferis R. Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action.Trends Pharmacol. Sci. 2009; 30: 356-362Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Herceptin, also known as Trastuzumab, marketed by Genentech Inc. is one example of therapeutic IgG1 antibody. It is produced from mammalian cell culture using Chinese hamster ovary cells (9Jefferis R. Glycosylation of recombinant antibody therapeutics.Biotechnol. Prog. 2005; 21: 11-16Crossref PubMed Scopus (436) Google Scholar). The main oligosaccharide forms found in this polypeptide chain in the Fc domain at asparagine 297 are biantennary core-fucosylated complex type structures with variable terminal galactosylation (zero, one, or two galactose residues) on their nonreducing termini (10Kamoda S. Nomura C. Kinoshita M. Nishiura S. Ishikawa R. Kakehi K. Kawasaki N. Hayakawa T. Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis.J Chromatogr A. 2004; 1050: 211-216Crossref PubMed Google Scholar, 11Jefferis R. Glycosylation of antibody therapeutics: optimisation for purpose.Methods Mol. Biol. 2009; 483: 223-238Crossref PubMed Scopus (34) Google Scholar). This humanized monoclonal antibody is known to effectively target breast cancer cells overexpresing the human epidermal growth factor receptor HER2/neu (12Slamon D.J. Leyland-Jones B. Shak S. Fuchs H. Paton V. Bajamonde A. Fleming T. Eiermann W. Wolter J. Pegram M. Baselga J. Norton L. Use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses her2.N. Engl. J. Med. 2001; 344: 783-792Crossref PubMed Scopus (9169) Google Scholar). HER2 is a cell membrane surface-bound receptor tyrosine kinase and is normally involved in the signal transduction pathways leading to cell growth and differentiation. It can be found overexpressed in a variety tumors' cells of epithelial origin and hematological malignancies, including acute lymphoblastic leukemia (13Müller M.R. Grünebach F. Kayser K. Vogel W. Nencioni A. Brugger W. Kanz L. Brossart P. Expression of her-2/neu on acute lymphoblastic leukemias: implications for the development of immunotherapeutic approaches.Clin. Cancer Res. 2003; 9: 3448-3453PubMed Google Scholar). When antibody binds to defective HER2 protein, this protein no longer causes cells to reproduce uncontrollably. This increases the survival of people with cancer. However, cancers usually develop resistance to trastuzumab. Unfortunately, only 25–30% of patients with HER2/neu positive breast cancer respond to this antibody (14Musolino A. Naldi N. Bortesi B. Pezzuolo D. Capelletti M. Missale G. Laccabue D. Zerbini A. Camisa R. Bisagni G. Neri T.M. Ardizzoni A. Immunoglobulin g fragment c receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with her-2/neu-positive metastatic breast cancer.J. Clin. Oncol. 2008; 26: 1789-1796Crossref PubMed Scopus (789) Google Scholar, 15Jones A. Combining trastuzumab (herceptin) with hormonal therapy in breast cancer: what can be expected and why?.Ann. Oncol. 2003; 14: 1697-1704Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 16Romond E.H. Perez E.A. Bryant J. Suman V.J. Geyer Jr, C.E. Davidson N.E. Tan-Chiu E. Martino S. Paik S. Kaufman P.A. Swain S.M. Pisansky T.M. Fehrenbacher L. Kutteh L.A. Vogel V.G. Visscher D.W. Yothers G. Jenkins R.B. Brown A.M. Dakhil S.R. Mamounas E.P. Lingle W.L. Klein P.M. Ingle J.N. Wolmark N. Trastuzumab plus adjuvant chemotherapy for operable her2-positive breast cancer.N. Engl. J. Med. 2005; 353: 1673-1684Crossref PubMed Scopus (4454) Google Scholar, 17Arnould L. Gelly M. Penault-Llorca F. Benoit L. Bonnetain F. Migeon C. Cabaret V. Fermeaux V. Bertheau P. Garnier J. Jeannin J.F. Coudert B. Trastuzumab-based treatment of her2-positive breast cancer: an antibody-dependent cellular cytotoxicity mechanism?.Br. J. Cancer. 2006; 94: 259-267Crossref PubMed Scopus (424) Google Scholar). Therefore search for the potential biomarkers that could predict the efficacy of clinical outcomes is needed. More precise investigation on cellular and molecular level might provide many exciting insights in understanding of mechanism resistance cancer cells to the antibody, so that antibody-based therapies can be optimized more individually (18Toi M. Horiguchi K. Bando H. Saji S. Chow L.W.C. Trastuzumab: updates and future issues.Cancer Chemother. Pharmacol. 2005; 56: 94-99Crossref PubMed Scopus (17) Google Scholar). We recently demonstrated how the carbohydrate moieties of two cancer cell models were affected during treatment with antibody (19Lattová E. Tomanek B. Bartusik D. Perreault H. N-glycomic changes in human breast carcinoma mcf-7 and t-lymphoblastoid cells after treatment with herceptin and herceptin/lipoplex.J. Proteome Res. 2010; 9: 1533-1540Crossref PubMed Scopus (38) Google Scholar). The detailed glycans profiles studied by means of mass spectrometry (MS) from the two most common cancer cell lines—human breast MCF-7 carcinoma and T-lymphoblastoid CEM cells before and after treatment with Herceptin showed significant differences. Dominant high-mannose structures in cancer cells were after treatment and complex and glycans were the structures found in the treated or with and of of the cancer cells to the antibody. The results for of glycosylation during In this on this has been to the investigation of glycosylated peptides by of the cancer cells before and after to Herceptin or analysis of glycopeptides by has been shown as one of the most and for a of It can provide on with of peptide K. M.R. C.E. and of an to Chem. 2001; PubMed Scopus Google Scholar, E. P. Perreault H. for investigation of oligosaccharides from of glycosylation in Chem. 2006; PubMed Scopus Google Scholar, E.P. Y. H. a for analysis using a Chem. 2007; PubMed Scopus Google Scholar, C. C. R. B. analysis of post-translational modifications in complex Proteome Res. 2009; PubMed Scopus Google Scholar, Y. A. A for of and and their on glycosylation Proteome Res. 2008; PubMed Scopus Google Scholar, B. M. R. H. T. of n-linked glycosylation on recombinant produced in using and Mol. Biol. 2009; Google Scholar, of for and implications for Proteome Res. 2009; PubMed Scopus Google Scholar, M. A.M. of glycosylated human peptides in from 2010; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, N.E. A.M. J. B. L. D. P. M.R. S.J. using and by and to the n-linked of and more biomarkers to changes in the post-translational modification at the during cancer or during The human breast carcinoma MCF-7 cell were obtained from the cells were from the cell samples were in a as previously described D. Tomanek B. E. Perreault H. G. treatment of human mcf-7 breast carcinoma with antibody, and using 2010; PubMed Scopus Google Scholar, D. Tomanek B. E. Perreault H. G. of cells and treated in the three 2010; PubMed Scopus Google Scholar). cells were at of and The culture for MCF-7 cells of with and and In of CEM the culture with and of cell were with The cells were from the in and their viability using the were with a When the cells a of cells treatment with Herceptin at or transfected with in the The before cells were in growth mixed with Herceptin to a of DNA in and to the to a DNA plasmid to of The mixed with for to the The with a of viability by at after The of with by and a protein to the after the presence of Herceptin, after treatment the were with a of during at with in of and in of for cells were with and by were by the T.M. P. C.E. of of human cancer in a PubMed Scopus Google Scholar). cells were in and with at for nonreducing or with in presence of for at and the in the at for the with and with at a of at for the the and the with samples were on a with a and a The with a The samples were with in as and abbreviations used epidermal growth factor mass and in membrane mass in as at a of from to at were and at human epidermal growth factor receptor immunoglobulin-γ mass spectrometry mass spectrometry and in membrane peptide plasmid DNA modification mass on a to a previously described E. V. R. Perreault H. of from of with 2009; PubMed Scopus Google Scholar). The with in of The the with and to The with of and the glycans into using were and after the with in of the with a and to The with of and the the The with of with of were from the three with of in and three with of were into using and samples were in of and of or a of and on the of a and were and were on the mass in the positive with a mass of and M. W. A mass with a and 2000; 14: PubMed Scopus Google Scholar). The using and a mass of were by for were for The as a function of the mass The using from The peptide were using a and using the of search with an on the to The were against the and for were for as with one and variable modifications were for of and from and were In for peptide were spectra of peptides in were by and P. J. for a common for in mass spectra of PubMed Scopus Google Scholar). The of oligosaccharide to the peptides on the produced and after and of glycans with E. P. Perreault H. for investigation of oligosaccharides from of glycosylation in Chem. 2006; PubMed Scopus Google E. Chen S. T. Perreault H. for the investigation of oligosaccharides and glycosylation in glycopeptides and 2007; 21: PubMed Scopus Google Scholar). of described previously were E. Perreault H. mass spectrometry and of of n-linked oligosaccharides from 2004; PubMed Scopus Google Scholar). of cell obtained before and after were to or on by as described in the Although the glycosylation of monoclonal IgG1 has been studied extensively and has been that monoclonal antibodies can in their and detailed analysis of is Chen W. M. J.R. H. mass spectrometry as a to the in of the therapeutic monoclonal antibody 2009; PubMed Scopus Google Scholar). the same of Herceptin used for treatment in a analysis of as previously (19Lattová E. Tomanek B. Bartusik D. Perreault H. N-glycomic changes in human breast carcinoma mcf-7 and t-lymphoblastoid cells after treatment with herceptin and herceptin/lipoplex.J. Proteome Res. 2010; 9: 1533-1540Crossref PubMed Scopus (38) Google Scholar). a detailed analysis of glycopeptides obtained from the antibody is with the investigation of glycosylated peptides isolated from treated to the profiles obtained from Herceptin were on a at a of in the spectra from are in peptides in the produced at and and to spectra their corresponded to a peptide bearing biantennary oligosaccharides with two and one The as the in the by a series of most of which corresponded to sialylated and oligosaccharides one and The glycopeptides detected in the were bearing characteristic biantennary glycans associated with IgG1 and were detected at and glycopeptides in the last were at and and their corresponded to high-mannose and complex structures with no galactose at the nonreducing glycopeptides in Herceptin were with a peptide of a characteristic with glycosylation at asparagine by the peptide from asparagine to confirmed by as fragment the and detected at and glycopeptides produced with peptide detected at In during analysis of the peptide with of confirmed E. P. Perreault H. for investigation of oligosaccharides from of glycosylation in Chem. 2006; PubMed Scopus Google Scholar). However, of and a of the fragment the spectra of glycopeptides detected from Herceptin were not for of glycopeptides in cancer cells after CEM cells treated with CEM cells transfected with MCF-7 cells treated with MCF-7 cells transfected with from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the from antibody used for the in a new The of N-glycans glycopeptides are in with the oligosaccharide obtained after from IgG1. approaches showed more in glycans than usually described in with IgG1. Although the of monoclonal antibodies are to be of J. M. D. R. F. of glycosylation by of glycopeptides and 2008; PubMed Scopus Google indicate of sialylated glycans in Herceptin, positive used for In the mass spectra from cancer cells treated with the antibody, to glycopeptides were in a of with the characteristic mass differences of and glycopeptides from MCF-7 and CEM cells after treatment with Herceptin or with are listed in I. The spectra from of CEM cell samples after Herceptin treatment are shown in and a obtained from Herceptin and the same the most glycopeptides were detected at and in all peptides plus at and including of sialylated glycopeptides in and were found in all treated produced The of moieties at The fragment detected at the presence of the the same as detected and in Herceptin The oligosaccharides corresponded to biantennary core-fucosylated In the same from treated the corresponded to of new glycosylated which were detected in the cells in the antibody the in at and were detected after of treatment in in and were associated with a peptide of However, of and of fragment in the of glycopeptides or their peptide after not to the of In the of CEM cells transfected with and with nonreducing were at and in the glycopeptides were with the mass of a peptide at and the same glycans as found in Herceptin at and were detected in from cells after of treatment are in in glycopeptides the same glycans as for the antibody. on the and characteristic the detected at could be to fragment to peptide and detected at and a peptide of or or The further at and in the for with after more in the In the spectra of cells treated with Herceptin significant of glycoconjugates were detected at and in The of glycopeptides were with the biantennary glycans as detected in and differed by for at the of and the associated with the of detected at The of at of at the to the The of produced a characteristic associated with a peptide at The fragment at and could be to from The of this peptide can be by a of fragment with a characteristic of The new glycopeptides not and to have not been described their associated with the of cancer cells to Herceptin treatment In used two cancer cell to viability of cells before and after treatment with Herceptin on different Although this of treatment not fully to treatment in can provide important molecular changes associated with In one of the cancer cells were treated with the antibody for This showed to be the In the the same cells were exposed to the antibody with Lipoplex and in this for the same cancer cells were more It is that a role in plasmid DNA into cell as as in of the membrane L. E.H. for Mol. 2000; Google and can more antibody of showed also differences at the The of N-glycans in cancer cells before and after to the antibody showed significant alterations in glycosylation (19Lattová E. Tomanek B. Bartusik D. Perreault H. N-glycomic changes in human breast carcinoma mcf-7 and t-lymphoblastoid cells after treatment with herceptin and herceptin/lipoplex.J. Proteome Res. 2010; 9: 1533-1540Crossref PubMed Scopus (38) Google Scholar). Dominant high-mannose glycans by of and sialylated glycans were found in cancer cells and after treatment structures were by complex biantennary oligosaccharides of the same as found in IgG1. A of significant oligosaccharide to nonfucosylated which were in the antibody in the cancer were found in cells treated only with Herceptin and were not detected when Herceptin with the of this on the of glycosylated peptides isolated from cancer cells after treatment and associated with the N-glycans It is expected that during antibody cancer cells are into in the cancer cells can be expected to be and IgG1 molecules be among and in treated expected glycopeptides in treated samples to be of the same as found in IgG1 in in their confirmed a characteristic peptide at with the the oligosaccharides detected in all treated cells corresponded to biantennary complex in the level of terminal forms of glycans as sialylated complex and structures such as high-mannose or were found in all treated The of glycans profiles obtained from samples analysis that obtained by from cell showed for of cancer cells which were transfected with In the of which cancer cells were exposed to Herceptin the glycopeptides analysis confirmed the same glycans as in However, of the peptides bearing of nonfucosylated biantennary and including sialylated structures were detected as after from the cell In on the investigation of oligosaccharides from glycopeptides shown that the of oligosaccharides were with of the glycopeptides E. P. Perreault H. for investigation of oligosaccharides from of glycosylation in Chem. 2006; PubMed Scopus Google Scholar). to profiles obtained from cancer cells after treatment with Herceptin by two different the of glycosylated peptides bearing nonfucosylated oligosaccharides could be by of peptides in the It is also that of glycopeptides were the mass for the Although the of the associated with glycans mass or are from than of which growth a of Herceptin treatment the presence of It be that of glycopeptides or glycans in treated cells were detected in MCF-7 and CEM It to that the spectra from new glycopeptides in all treated cells only for the glycans of the same as found in differed in peptide the most significant corresponded to glycoconjugates with peptides and and in glycopeptides detected at and from CEM cells after with have been previously in the of Fc domain of affected by A.J. E.H. R. J. of of a complex by terminal is in and 2007; 17: PubMed Scopus Google Scholar). to all detected including with not fully showed as a of their as found in IgG1 and were bearing the same type glycans of the same profiles as were in the antibody. and the of the on two different cancer cell models that all of new glycosylated peptides described are results of transformation in the IgG1 during have previously of cancer cells with the viability of cells to a than treatment with Herceptin D. Tomanek B. E. Perreault H. G. treatment of human mcf-7 breast carcinoma with antibody, and using 2010; PubMed Scopus Google Scholar). Lipofectamine is known to the membrane remains how the of Herceptin. Although further are to pathways that alterations in in the have shown that more new glycopeptides were found in cancer cells treated with Herceptin than in cells exposed to The of the peptides and the structures indicate that described glycopeptides might be the of of Herceptin. Therefore is to in the with Lipoplex results in of Herceptin, of the complex to different membrane or a in the of Herceptin. results indicate that presence of could a role not only in better also prevents antibody from and elongates its proper function during In the results in this indicate that treatment is more complex and series pathways on different molecular The precise and better understanding of changes in glycoconjugates might provide new how to treatment monoclonal antibody. We and of the of for the and We have no with

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