Discovery of Pancreatic Ductal Adenocarcinoma-Related Aberrant Glycosylations: A Multilateral Approach of Lectin Microarray-Based Tissue Glycomic Profiling With Public Transcriptomic Datasets

Abstract

Aberrant protein glycosylation is one of the most notable features in cancerous tissues, and thereby glycoproteins with disease-relevant glycosylation alterations are fascinating targets for the development of biomarkers and therapeutic agents. For this purpose, a reliable strategy is needed for the analysis of glycosylation alterations occurring on specific glycoproteins during the progression of cancer. Here, we propose a bilateral approach combining lectin microarray-based tissue glycomic profiling and database-derived transcriptomic datasets. First, lectin microarray was used to perform differential glycomic profiling of crude extracts derived from non-tumor and tumor regions of frozen tissue sections from pancreatic ductal adenocarcinoma (PDAC). This analysis revealed two notable tissue glycome alterations in PDAC samples: increases in sialylated glycans and bisecting N-acetylglucosamine and a decrease in ABO blood group antigens. To examine aberrations in the glycosylation machinery related to these glycomic alterations, we next employed public datasets of gene expression profiles in cancerous and normal pancreases provided by The Cancer Genome Atlas and the Genotype-Tissue Expression projects, respectively. In this analysis, glycosyltransferases responsible for the glycosylation alterations showed aberrant gene expression in the cancerous tissues, consistent with the tissue glycomic profiles. The correlated alterations in glycosyltransferase expression and tissue glycomics were then evaluated by differential glycan profiling of a membrane N-glycoprotein, basigin, expressed in tumor and non-tumor pancreatic cells. The focused differential glycomic profiling for endogenous basigin derived from non-tumor and cancerous regions of PDAC tissue sections demonstrated that PDAC-relevant glycan alterations of basigin closely reflected the notable features in the disease-specific alterations in the tissue glycomes. In conclusion, the present multi-omics strategy using public transcriptomic datasets and experimental glycomic profiling using a tiny amount of clinical specimens successfully demonstrated that basigin is a representative N-glycoprotein that reflects PDAC-related aberrant glycosylations. This study indicates the usefulness of large public data sets such as the gene expression profiles of glycosylation-related genes for evaluation of the highly sensitive tissue glycomic profiling results. This strategy is expected to be useful for the discovery of novel glyco-biomarkers and glyco-therapeutic targets.

Keywords: ABO blood group antigen; Genotype-Tissue Expression (GTEx); The Cancer Genome Atlas (TCGA); basigin/CD147; glycosylation; glycosyltransferase; lectin microarray; multi-omics.

Figure 1

Differential glycomic profiling of PDAC tissue sections. (A) Representative images of non-tumor and tumor regions in hematoxylin and eosin-stained frozen sections. The arrows indicate the cancerous ducts. (B) Schematic overview of lectin microarray-based glycomic profiling of unstained frozen sections.

Figure 2

Univariate analysis of the tissue glycomic profiles obtained from the non-tumor (N) and tumor (T) regions of PDAC tissue sections. (A) Comparison of the relative signal intensities of the five lectins that showed extremely significant higher signals (P = 0.0001). (B) Comparison of the relative signal intensities of the seven lectins recognizing ABO blood group antigens. N = 14 for each region. *P < 0.05, **P = 0.0001. The median and P-values are summarized in Table 1.

Figure 3

Multivariate analysis of the tissue glycomic profiles by PCA. In the score plot (left panel), the lectin microarray data of the non-tumor (blue) and tumor (red) regions of PDAC tissue sections (N = 14) were plotted. The numbers of three non-tumor samples obtained from O blood group patients (No. 11–13) are indicated. In the loading plot (right panel), the relative signal intensities of 45 lectins on the array are plotted. The lectins extracted for the non-tumor and tumor regions are indicated in blue and red, respectively.

Figure 4

Comparison of the gene expression levels of selected glycosyltransferases between the normal (Np) and cancerous (Cp) pancreases. (A) Glycan structures of the O, A, and B blood group antigens (upper panel) and gene expression levels of their related glycosyltransferases (lower panels). (B) Gene expression levels of sialyltransferases related to α2,3-, and α2,6-sialylation of terminal galactose residues. (C) Gene expression levels of mannosyl-glycoprotein N-acetylglucosaminyltransferases related to the biosynthesis of bisecting GlcNAc. N = 165 for Np and N = 178 for Cp. **P = 0.0001.

Figure 5

Expression analyses of endogenous basigin in PDAC tissues. (A) Expression levels of basigin-encoding gene (BSG) in normal (Np; N = 165) and cancerous (Cp; N = 178) pancreases. **P = 0.0001. (B) Representative images of the non-tumor (upper panel) and tumor (lower panel) regions of FFPE sections stained for basigin. The arrow indicates the islet of Langerhans, where basigin was not expressed. IHC, immunohistochemistry. (C) Western blot analysis for basigin immunoprecipitated from frozen tissues of representative PDAC cases. This N-glycoprotein was detected as high- and low-glycosylated forms. IP, immunoprecipitation; IB, immunoblot.

Figure 6

Univariate analysis of the glycan profiles of basigin derived from the non-tumor (N) and tumor (T) regions of PDAC tissue sections. (A) Comparison of the relative signal intensities of the six lectins that showed extremely significant differences (P = 0.0001) in the differential glycan profiling of basigin and/or in the differential tissue glycomic profiling (Figure 2A). (B) Comparison of the relative signal intensities of the seven lectins recognizing ABO blood group antigens. N = 14 for each region. *P < 0.05, **P = 0.0001. The median and P-values are summarized in Table 2.

Figure 7

Multivariate analysis of the glycan profiles of basigin by PCA. In the score plot (left panel), the lectin microarray data of endogenous basigin enriched from the non-tumor (blue) and tumor (red) regions of PDAC tissue sections (N = 14) are plotted. The numbers of three non-tumor samples obtained from O blood group patients (No. 11–13) are indicated. In the loading plot (right panel), the relative signal intensities of 45 lectins on the array are plotted. The lectins extracted from the non-tumor and tumor regions are indicated in blue and red, respectively.