Comprehensive characterization of complex glycosphingolipids in human pancreatic cancer tissues
2023; Elsevier BV; Volume: 299; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2023.102923
ISSN1083-351X
AutoresKarel Hořejší, Chunsheng Jin, Zuzana Vaňková, Robert Jirásko, Ondřej Strouhal, Bohuslav Melichar, Susann Teneberg, Michal Holčapek,
Tópico(s)Ubiquitin and proteasome pathways
ResumoPancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of TLC, chemical staining, carbohydrate-recognized ligand-binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4) and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches. Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes of cancer-related deaths worldwide, accounting for 90% of primary pancreatic tumors with an average 5-year survival rate of less than 10%. PDAC exhibits aggressive biology, which, together with late detection, results in most PDAC patients presenting with unresectable, locally advanced, or metastatic disease. In-depth lipid profiling and screening of potential biomarkers currently appear to be a promising approach for early detection of PDAC or other cancers. Here, we isolated and characterized complex glycosphingolipids (GSL) from normal and tumor pancreatic tissues of patients with PDAC using a combination of TLC, chemical staining, carbohydrate-recognized ligand-binding assay, and LC/ESI-MS2. The major neutral GSL identified were GSL with the terminal blood groups A, B, H, Lea, Leb, Lex, Ley, P1, and PX2 determinants together with globo- (Gb3 and Gb4) and neolacto-series GSL (nLc4 and nLc6). We also revealed that the neutral GSL profiles and their relative amounts differ between normal and tumor tissues. Additionally, the normal and tumor pancreatic tissues differ in type 1/2 core chains. Sulfatides and GM3 gangliosides were the predominant acidic GSL along with the minor sialyl-nLc4/nLc6 and sialyl-Lea/Lex. The comprehensive analysis of GSL in human PDAC tissues extends the GSL coverage and provides an important platform for further studies of GSL alterations; therefore, it could contribute to the development of new biomarkers and therapeutic approaches. Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent type of primary pancreatic malignant tumors (accounting for more than 90% of all types of pancreatic cancer) with highly aggressive behavior and extremely poor prognosis (1Schawkat K. Manning M.A. Glickman J.N. Mortele K.J. Pancreatic ductal adenocarcinoma and its variants: pearls and perils.Radiographics. 2020; 40: 1219-1239Crossref PubMed Scopus (33) Google Scholar, 2Orth M. Metzger P. Gerum S. Mayerle J. Schneider G. Belka C. et al.Pancreatic ductal adenocarcinoma: biological hallmarks, current status, and future perspectives of combined modality treatment approaches.Radiat. Oncol. 2019; 14: 1-20Crossref PubMed Scopus (224) Google Scholar, 3Sarantis P. Koustas E. 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Another significant hallmark of PDAC is high resistance and low response rate to treatment with anticancer drugs and radiation (1Schawkat K. Manning M.A. Glickman J.N. Mortele K.J. Pancreatic ductal adenocarcinoma and its variants: pearls and perils.Radiographics. 2020; 40: 1219-1239Crossref PubMed Scopus (33) Google Scholar, 2Orth M. Metzger P. Gerum S. Mayerle J. Schneider G. Belka C. et al.Pancreatic ductal adenocarcinoma: biological hallmarks, current status, and future perspectives of combined modality treatment approaches.Radiat. Oncol. 2019; 14: 1-20Crossref PubMed Scopus (224) Google Scholar, 5Mahajan U.M. Alnatsha A. Li Q. Oehrle B. Weiss F.U. Sendler M. et al.Plasma metabolome profiling identifies metabolic subtypes of pancreatic ductal adenocarcinoma.Cells. 2021; 10: 1-16Crossref Scopus (7) Google Scholar). The high resistance of PDAC to available therapies, together with late detection, results in a 5-year overall survival rate of less than 10% and, particularly in metastatic PDAC, an overall 1-year survival rate of less than 20%. This makes PDAC the most lethal cancer (1Schawkat K. Manning M.A. Glickman J.N. Mortele K.J. Pancreatic ductal adenocarcinoma and its variants: pearls and perils.Radiographics. 2020; 40: 1219-1239Crossref PubMed Scopus (33) Google Scholar, 2Orth M. Metzger P. Gerum S. Mayerle J. Schneider G. Belka C. et al.Pancreatic ductal adenocarcinoma: biological hallmarks, current status, and future perspectives of combined modality treatment approaches.Radiat. Oncol. 2019; 14: 1-20Crossref PubMed Scopus (224) Google Scholar, 3Sarantis P. Koustas E. Papadimitropoulou A. Papavassiliou A.G. Karamouzis M.V. Pancreatic ductal adenocarcinoma: treatment hurdles, tumor microenvironment and immunotherapy.World J. Gastrointest. 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The carbohydrate antigen sialyl Lewisa (i.e., sLea or CA 19-9) is one of the well-known and frequently used serological biomarkers for the clinical diagnosis of pancreatic (7Shida K. Korekane H. Misonou Y. Noura S. Ohue M. Takahashi H. et al.Novel ganglioside found in adenocarcinoma cells of Lewis-negative patients.Glycobiology. 2010; 20: 1594-1606Crossref PubMed Scopus (10) Google Scholar, 8Mayerle J. Kalthoff H. Reszka R. Kamlage B. Peter E. Schniewind B. et al.Metabolic biomarker signature to differentiate pancreatic ductal adenocarcinoma from chronic pancreatitis.Gut. 2018; 67: 128-137Crossref PubMed Scopus (180) Google Scholar), gastrointestinal, and other types of epithelial cancers (9Furukawa K. Ohmi Y. Ohkawa Y. Bhuiyan R.H. Zhang P. Tajima O. et al.New era of research on cancer-associated glycosphingolipids.Cancer Sci. 2019; 110: 1544-1551Crossref PubMed Scopus (52) Google Scholar). 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Zhang J. et al.Differential O- and glycosphingolipid glycosylation in human pancreatic adenocarcinoma cells with opposite morphology and metastatic behavior.Front. Oncol. 2020; 10: 1-19PubMed Google Scholar) demonstrated that GSL-glycosylation and O-glycosylation play a more dominant role, in particular in pancreatic cancer, than N-glycosylation (46Holst S. Belo A.I. Giovannetti E. Van Die I. Wuhrer M. Profiling of different pancreatic cancer cells used as models for metastatic behaviour shows large variation in their N-glycosylation.Sci. Rep. 2017; 7: 16623Crossref PubMed Scopus (40) Google Scholar). However, targeted approaches that focus mainly on tumor cells and predefined metabolic pathways may not show the full extent of complex metabolic alterations (5Mahajan U.M. Alnatsha A. Li Q. Oehrle B. Weiss F.U. Sendler M. et al.Plasma metabolome profiling identifies metabolic subtypes of pancreatic ductal adenocarcinoma.Cells. 2021; 10: 1-16Crossref Scopus (7) Google Scholar). In addition, there are still major challenges that stem mainly from the lack of sensitive, accurate, and reliable methods for the separation of GSL isomers as well as for the detection, identification, and quantitation of less prevalent GSL species (47Barrientos R.C. Zhang Q. Recent advances in the mass spectrometric analysis of glycosphingolipidome – a review.Anal. Chim. Acta. 2020; 1132: 134-155Crossref PubMed Scopus (17) Google Scholar). The aim of the present study is to characterize the GSL of human pancreatic tissues of patients with PDAC with a particular interest in minor complex GSL to expand the database of lipids that are routinely analyzed and to allow mutual comparison of GSL alterations in normal and tumor pancreatic tissues. The future perspective of this study is to incorporate these complex GSL into the screening method for PDAC based on body fluid analysis, as recently published by our research group (14Wolrab D. Jirásko R. Cífková E. Höring M. Mei D. Chocholoušková M. et al.Lipidomic profiling of human serum enables detection of pancreatic cancer.Nat. Commun. 2022; 13: 1-16Crossref PubMed Scopus (53) Google Scholar). The GSL were isolated by a micro method (Fig. 1) according to Barone et al. (48Barone A. Benktander J. Teneberg S. Breimer M.E. Characterization of acid and non-acid glycosphingolipids of porcine heart valve cusps as potential immune targets in biological heart valve grafts.Xenotransplantation. 2014; 21: 510-522Crossref PubMed Scopus (27) Google Scholar), which allows the isolation and purification of GSL with a wider range of carbohydrate units. This is of particular advantage for complex GSL that are found in biological materials in tiny amounts, and their effective isolation by conventional extraction methods, such as Folch (49Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), Bligh and Dyer (50Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (44558) Google Scholar), or Matyash (51Matyash V. Liebisch G. Kurzchalia T.V. Shevchenko A. Schwudke D. Lipid extraction by methyl-terf-butyl ether for high-throughput lipidomics.J. Lipid Res. 2008; 49: 1137-1146Abstract Full Text Full Text PDF PubMed Scopus (1554) Google Scholar), has not yet been described. In total, 24 paired tissue samples of tumor and normal tissues were collected from 12 patients. After total lipid extraction, the extracts were subjected to mild alkaline methanolysis to remove acylglycerols and alkali-labile phospholipids. The purpose of the ensuing acetylation was to change the polarity of glycolipids from polar to nonpolar so that alkali-stable phospholipids (mainly sphingomyelins) were removed. Consequently, acetylated GSL were separated from the nonpolar compounds (e.g., ceramides) and alkali-stable phospholipids (especially sphingomyelins) using silica-based chromatography. After deacetylation, the GSL were separated into neutral GSL (N-GSL) and acid GSL (A-GSL) fractions using ion-exchange chromatography. In summary, 6.3 mg and 26.2 mg of N-GSL were obtained, together with 11.6 mg and 14.3 mg of A-GSL from pooled tumor and normal pancreatic tissues, respectively (Table 1).Table 1Amounts of acid and neutral glycosphingolipids obtained from normal and tumor pancreatic tissues of PDAC patients and expressed in mg of glycosphingolipids per g of tissues in dry weightType of sampleWet weight[g]Dry weight[g]N-GSL[mg]N-GSL[mg/g tissue]A-GSL[mg]A-GSL[mg/g tissue]Pooled tissues; T1.0890.6066.310.411.619.1Pooled tissues; N2.0921.23226.221.314.311.6N-GSL and A-GSL denote total neutral and acid glycosphingolipids, respectively. T and N denote tumor and normal, respectively, and ND denotes not determined. Open table in a new tab N-GSL and A-GSL denote total neutral and acid glycosphingolipids, respectively. T and N denote tumor and normal, respectively, and ND denotes not determined. Rhodococcus spp. recombinant endoglycoceramidase II (rEGCase II) was used for the hydrolysis of GSL, although the hydrolytic capacity of this enzyme to globo-series GSL and some gangliosides is restricted (28Albrecht S. Vainauskas S. Stöckmann H. McManus C. Taron C.H. Rudd P.M. Comprehensive profiling of glycosphingolipid glycans using a novel broad specificity endoglycoceramidase in a high-throughput workflow.Anal. Chem. 2016; 88: 4795-4802Crossref PubMed Scopus (32) Google Scholar). In contrast, EGCase I has a broader substrate specificity and better reaction efficiency than EGCase II and III (52Ishibashi Y. Kobayashi U. Hijikata A. Sakaguchi K. Goda H.M. Tamura T. et al.Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system.J. Lipid Res. 2012; 53: 2242-2251Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 53Burla B. Arita M. Arita M. Bendt A.K. Cazenave-Gassiot A. Dennis E.A. et al.MS-based lipidomics of human blood plasma: a community-initiated position paper to develop accepted guidelines.J. Lipid Res. 2018; 59: 2001-2017Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). However, the use of rEGCase II in this study was intentional because globotriaosylceramide and globotetraosylceramide (Gb3 and Gb4) are major GSL of many tissues, resulting in MS spectra being dominated by Gb3 and Gb4 ions. The main advantage of using rEGCase II in this study is that it allowed the detection of low abundant complex GSL. We performed liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS2) analysi
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