Gastric Cancer Cell Glycosylation as a Modulator of the ErbB2 Oncogenic Receptor

International Journal of Molecular Sciences Article Gastric Cancer Cell Glycosylation as a Modulator of the ErbB2 Oncogenic Receptor Henrique O. Duarte 1,2,3 ID , Meritxell Balmaña 1,2, Stefan Mereiter 1,2, Hugo Osório 1,2,4, Joana Gomes 1,2,*ID and Celso A. Reis 1,2,3,4,* 1Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal; [email protected] (H.O.D.); [email protected] (M.B.); [email protected] (S.M.); [email protected] (H.O.) 2Institute of Molecular Pathology and Immunology, University of Porto, 4200-135 Porto, Portugal 3Institute of Biomedical Sciences Abel Salazar, University of Porto, 4050-313 Porto, Portugal 4Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal *Correspondence: [email protected] (J.G.); [email protected] (C.A.R.); Tel.: +351-22-040-88-00 (ext. 6067) (J.G.); +351-22-040-88-00 (ext. 6068) (C.A.R.) Received: 28 September 2017; Accepted: 25 October 2017; Published: 28 October 2017 Abstract: Aberrant expression and hyperactivation of the human epidermal growth factor receptor 2 (ErbB2) constitute crucial molecular events underpinning gastric neoplastic transformation. Despite ErbB2 extracellular domain being a well-known target for glycosylation, its glycosylation profile and the molecular mechanisms through which it actively tunes tumorigenesis in gastric cancer (GC) cells remain elusive. We aimed at disclosing relevant ErbB2 glycan signatures and their functional impact on receptor’s biology in GC cells. The transcriptomic profile of cancer-relevant glycosylation enzymes, and the expression and activation of the ErbB receptors were characterized in four GC cell lines. Cellularand receptor-specific glycan profiling of ErbB2-overexpressing NCI-N87 cells unveiled a heterogeneous glycosylation pattern harboring the tumor-associated sialyl Lewis a (SLe a ) antigen. The expression of SLe a and key enzymes integrating its biosynthetic pathway were strongly upregulated in this GC cell line. An association between the expression of ERBB2 and FUT3, a central gene in SLe a biosynthesis, was disclosed in GC patients, further highlighting the crosstalk between ErbB2 and SLe a expression. Moreover, cellular deglycosylation and CA 19.9 antibody-mediated blocking of SLe a drastically altered ErbB2 expression and activation in NCI-N87 cells. Altogether, NCI-N87 cell line constitutes an appealing in vitro model to address glycan-mediated regulation of ErbB2 in GC. Keywords: human epidermal growth factor receptor 2 (ErbB2); gastric cancer (GC); glycosylation; sialyl Lewis a (SLea); CA 19.9 1. Introduction Gastric cancer (GC) represents the fifth most incident human malignancy and the third leading cause of cancer-related deaths worldwide [ 1 ]. Being frequently diagnosed at advanced stages, at which surgical resection and conventional chemotherapeutic approaches fall short in improving patients’ dismal prognosis, GC remains a globally heavy health burden [2]. Overexpression of the human epidermal growth factor receptor 2 (ErbB2), a direct consequence of the ERBB2 gene amplification, constitutes a well-established molecular hallmark of multiple human solid tumors, and has been consistently reported as a key molecular driver of gastric carcinogenesis, by triggering the aberrant activation of complex downstream signaling pathways that ultimately dictate malignant cell behavior (reviewed in [ 3 ]). Indeed, ErbB2 overexpression is observed in 7% to 34% of GC patients and constitutes a predictive factor of poor disease prognosis [ 4 ]. The 185-kDa Int. J. Mol. Sci. 2017,18, 2262; doi:10.3390/ijms18112262 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017,18, 2262 2 of 19 ErbB2 transmembrane protein belongs to the ErbB epidermal growth factor receptor family of receptor tyrosine kinases (RTKs). Three additional members are included in this family (ErbB1/epidermal growth factor receptor (EGFR), ErbB3 and ErbB4), with which ErbB2 shares structural homology. The ErbB receptors are composed of an extracellular ligand-binding domain, a short hydrophobic transmembrane domain and cytosolic tail with intrinsic tyrosine kinase activity (only absent in ErbB3) [ 5 ]. By exhibiting a ligand-independent mode of activation and constituting the preferred heterodimerization partner of the remaining ErbB receptors, ErbB2 holds the most pronounced ability for potent and prolonged signal transduction. ErbB2 therefore carries an exponentially aggravated oncogenic and transforming potential and represents an excellent molecular candidate for selective anti-cancer targeted therapy [ 6 , 7 ]. In fact, the trastuzumab monoclonal antibody (MAb), targeting the subdomain IV of the ErbB2 extracellular region, has become the first personalized standard of care for patients harboring advanced ErbB2-positive GC, in combination with conventional chemotherapy [ 8 ]. However, the emergence of acquired tumor resistance, through still elusive molecular mechanisms, has limited the expected benefits from this therapeutic strategy [9,10]. Protein glycosylation is the enzymatic addition of sugar moieties to specific amino acid residues, and consists of a complex and finely regulated cellular process, representing the most abundant type of protein posttranslational modifications [ 11 , 12 ]. Glycosylation results from the well-orchestrated action of a wide range of enzymes and organelles and is acknowledged to be crucial in several cellular, physiological and pathological processes, including the carcinogenesis of multiple organs, such as the stomach [ 12 , 13 ]. The abnormal expression and activity of glycosyltransferase and glycosidase enzymes, as well as alterations in the structure and expression levels of cancer-associated glycan epitopes, have been consistently linked to the dismal prognosis of cancer patients (reviewed in [ 12 ]). Despite the remarkable advances made in glycan-based analytical techniques, many of the molecular mechanisms through which aberrant glycosylation actively tunes malignant cell behavior within the intricate context of tumor biology are still unknown [14]. RTK maturation is tightly regulated by protein N-linked glycosylation [ 15 ]. Furthermore, the glycosylation status of these transmembrane proteins has been shown to play a pivotal role in several aspects of receptor’s biology in the context of distinct human developmental and cancer models [16–20] . In particular, the extracellular domain of the ErbB2 receptor harbors seven putative sites for the anchorage of N-linked glycan structures (www.cbs.dtu.dk/NetNGlyc), five of which had their glycosylated status demonstrated in human mammary carcinoma cell lines [ 21 , 22 ]. However, protein glycosylation is highly context-dependent, which severely limits the extrapolation of relevant findings from one pathological setting to another [ 12 ]. In this sense, the detailed glycosylation profile of this oncogenic RTK within the context of GC requires further elucidation. In the present study, we have performed a molecular characterization of four GC cell lines (NCI-N87, AGS, MKN45 and MKN74), including the transcription analysis of cancer-relevant glycosylation enzymes, as well as the expression and activation status of the ErbB family of receptors. Additional cellular and receptor-specific characterization of NCI-N87, an in vitro model of ErbB2 overexpression and hyperactivation, unveiled a highly heterogeneous and complex glycosylation pattern encompassing well-established tumor-associated glycan determinants, such as the endothelial selectin-ligand sialyl Lewis a (SLe a ). The consistent expression of this glycan epitope and key enzymes of its biosynthetic pathway observed in ErbB2-overexpressing GC cells was further corroborated by in silico analysis. Moreover, an in silico association between the ERBB2 and FUT3 genes was disclosed in a cohort of GC patients. Finally, in vitro deglycosylation and antibody-mediated glycan-blocking of SLe a drastically altered receptor’s expression and activation in ErbB2-overexpressing NCI-N87 GC cells. Altogether, these results portray the NCI-N87 cell line as a useful in vitro tool to study the regulatory role of glycans in ErbB2-driven gastric carcinogenesis. Int. J. Mol. Sci. 2017,18, 2262 3 of 19 2. Results 2.1. Expression and Activation of the Human Epidermal Growth Factor Receptor Family in Gastric Cancer Cell Lines To characterize the expression pattern of the four RTKs integrating the ErbB family (ErbB1/EGFR, ErbB2, ErbB3 and ErbB4), Western blot (WB) analysis was performed on whole cell lysates from four GC cell lines (NCI-N87, AGS, MKN45 and MKN74), representative for the heterogeneous nature of gastric cancer regarding ErbB2 status (Figure 1). Furthermore, the phosphorylation status of the tyrosine-kinase domains of ErbB2 and EGFR receptors was additionally determined. In general, the four GC cell lines exhibited heterogeneous expression and activation of the analyzed RTKs. The ERBB2-amplified and well-differentiated NCI-N87 cell line displayed the highest levels of total ErbB2 protein, and was the only cell line with strong constitutive activation of this receptor (Figure 1a). ErbB2 expression was also detected in the remaining three GC cell lines, although in considerably lower levels. Despite similar levels of total EGFR expression being observed for NCI-N87 and MKN74, the former cell line displayed substantially higher receptor phosphorylation (Figure 1b). Although in substantially lower levels, endogenous EGFR activation was also detected in AGS, MKN45 and MKN74 cell lines. As for ErbB3, all cell lines displayed comparable protein levels of the receptor, with the exception of AGS where ErbB3 expression was lower (Figure 1c). Expression of ErbB4 was particularly low across the four screened GC cell lines. Int. J. Mol. Sci. 2017, 18, 2262 3 of 18 2. Results 2.1. Expression and Activation of the Human Epidermal Growth Factor Receptor Family in Gastric Cancer Cell Lines To characterize the expression pattern of the four RTKs integrating the ErbB family (ErbB1/EGFR, ErbB2, ErbB3 and ErbB4), Western blot (WB) analysis was performed on whole cell lysates from four GC cell lines (NCI-N87, AGS, MKN45 and MKN74), representative for the heterogeneous nature of gastric cancer regarding ErbB2 status (Figure 1). Furthermore, the phosphorylation status of the tyrosine-kinase domains of ErbB2 and EGFR receptors was additionally determined. In general, the four GC cell lines exhibited heterogeneous expression and activation of the analyzed RTKs. The ERBB2-amplified and well-differentiated NCI-N87 cell line displayed the highest levels of total ErbB2 protein, and was the only cell line with strong constitutive activation of this receptor (Figure 1a). ErbB2 expression was also detected in the remaining three GC cell lines, although in considerably lower levels. Despite similar levels of total EGFR expression being observed for NCI-N87 and MKN74, the former cell line displayed substantially higher receptor phosphorylation (Figure 1b). Although in substantially lower levels, endogenous EGFR activation was also detected in AGS, MKN45 and MKN74 cell lines. As for ErbB3, all cell lines displayed comparable protein levels of the receptor, with the exception of AGS where ErbB3 expression was lower (Figure 1c). Expression of ErbB4 was particularly low across the four screened GC cell lines. Figure 1. Western blot (WB) analysis of the total expression and activation (phosphorylated receptor) of the human epidermal growth factor receptor family members: (a) total and phosphorylated human epidermal growth factor receptor 2 (ErbB2 and pErbB2); (b) total and phosphorylated human epidermal growth factor receptor (EGFR and pEGFR); and (c) ErbB3 and ErbB4 in four gastric adenocarcinoma cell lines (NCI-N87, AGS, MKN45 and MKN74). Two distinct exposure times (a shorter and a longer one, from top to bottom, respectively) are depicted for the ErbB2 WB. α-Tubulin was used as a loading control. Band densities, normalized for the loading control (for total ErbB2 and EGFR) or both the loading control and total receptor (for pErbB2 and pEGFR), are depicted. All experiments were performed in triplicate and a representative WB analysis is depicted. 2.2. Glycosyltransferase Expression in Gastric Cancer Cell Lines The presence of tumor-associated glycan epitopes at the surface of malignant cells is regulated by the coordinated expression and activity of specific glycosyltransferases. In this sense, prior to an ErbB2-targeted glycosylation analysis, we first evaluated the general glycophenotype of the four GC cell lines. The transcript levels of eight key genes encoding for glycosyltransferases previously reported to be implicated in human neoplastic transformation were assessed by real-time quantitative PCR (RT-qPCR) (Figure 2). The FUT3 and FUT8 genes encode for enzymes with fucosyltransferase Figure 1. Western blot (WB) analysis of the total expression and activation (phosphorylated receptor) of the human epidermal growth factor receptor family members: ( a ) total and phosphorylated human epidermal growth factor receptor 2 (ErbB2 and pErbB2); ( b ) total and phosphorylated human epidermal growth factor receptor (EGFR and pEGFR); and ( c ) ErbB3 and ErbB4 in four gastric adenocarcinoma cell lines (NCI-N87, AGS, MKN45 and MKN74). Two distinct exposure times (a shorter and a longer one, from top to bottom, respectively) are depicted for the ErbB2 WB. α -Tubulin was used as a loading control. Band densities, normalized for the loading control (for total ErbB2 and EGFR) or both the loading control and total receptor (for pErbB2 and pEGFR), are depicted. All experiments were performed in triplicate and a representative WB analysis is depicted. 2.2. Glycosyltransferase Expression in Gastric Cancer Cell Lines The presence of tumor-associated glycan epitopes at the surface of malignant cells is regulated by the coordinated expression and activity of specific glycosyltransferases. In this sense, prior to an ErbB2-targeted glycosylation analysis, we first evaluated the general glycophenotype of the four GC cell lines. The transcript levels of eight key genes encoding for glycosyltransferases previously reported to be implicated in human neoplastic transformation were assessed by real-time quantitative PCR Int. J. Mol. Sci. 2017,18, 2262 4 of 19 (RT-qPCR) (Figure 2). The FUT3 and FUT8 genes encode for enzymes with fucosyltransferase activity responsible for the terminal ( α 1,3-linked) or core ( α 1,6-linked) fucosylation of their glycoconjugate targets, respectively. The MGAT3 and MGAT5 genes encode for enzymes that catalyze the addition of bisecting and branching N-acetylglucosamine (GlcNAc) during the biosynthesis of complex N-glycans, respectively. The ST3GAL3 and ST3GAL4 genes encode for sialyltransferases capable of adding α 2,3-linked sialic acids to the terminal non-reducing end of glycan chains. The ST6GAL1 gene encodes for an enzyme capable of transferring an α 2,6-linked sialic acid monosaccharide to N-glycan substrates. Finally, The B3GALT5 gene encodes for an enzyme involved in the biosynthesis of type 1 chains of oligosaccharides, which is prerequisite for the formation of Lewis a (Le a ), Lewis b (Le b ) and SLeaantigens. Int. J. Mol. Sci. 2017, 18, 2262 4 of 18 activity responsible for the terminal (α1,3-linked) or core (α1,6-linked) fucosylation of their glycoconjugate targets, respectively. The MGAT3 and MGAT5 genes encode for enzymes that catalyze the addition of bisecting and branching N-acetylglucosamine (GlcNAc) during the biosynthesis of complex N-glycans, respectively. The ST3GAL3 and ST3GAL4 genes encode for sialyltransferases capable of adding α2,3-linked sialic acids to the terminal non-reducing end of glycan chains. The ST6GAL1 gene encodes for an enzyme capable of transferring an α2,6-linked sialic acid monosaccharide to N-glycan substrates. Finally, The B3GALT5 gene encodes for an enzyme involved in the biosynthesis of type 1 chains of oligosaccharides, which is prerequisite for the formation of Lewis a (Lea), Lewis b (Leb) and SLea antigens. Figure 2. Real-time quantitative PCR (RT-qPCR) analysis of the relative transcript expression levels of eight cancer-relevant glycosyltransferase-coding genes (FUT3, FUT8, MGAT3, MGAT5, ST3GAL3, ST3GAL4, ST6GAL1, and B3GALT5) in four GC cell lines (NCI-N87, AGS, MKN45 and MKN74). For each cell line, the mRNA levels of a given gene are presented as 2−∆ΔCt. Target gene relative abundance was normalized to the mRNA levels of the RNA18S5 endogenous control. The obtained RT-qPCR uncovered a distinct glycosyltransferase expression signature for each of the screened GC cell lines. In particular, the B3GALT5 gene showed an expression range of multiple orders of magnitude across the four screened cell lines. Indeed, the diffuse type MKN45 cell line exhibits remarkably higher levels of this enzyme’s mRNA when compared to the other cell lines, especially in the case of intestinal type MKN74 cells where no mRNA was detected. A similar expression pattern was observed for ST6GAL1. However, regarding ST6GAL1, a generally higher transcript expression was consistently observed across the four screened GC cell lines, with the exception of MKN74, in comparison to the remaining screened glyco-genes. Illustrative of the cell line-specific glycosyltransferase expression signature, the ErbB2-overexpressing NCI-N87 cell line displayed the highest expression of FUT3, MGAT5 and ST3GAL4 genes, but also the lowest detected transcript levels of both FUT8 and ST3GAL3. The obtained results are in accordance with previous data published by our group, in respect to the GC cell lines and glycosyltransferase-coding genes commonly analyzed in both studies [23]. 2.3. Glycosylation Profile of ErbB2-Overexpressing NCI-N87 Cells Since the NCI-N87 GC cell line exhibited the highest levels of both ErbB2 expression and activation, it was therefore selected for further glycan epitope characterization. Due to the subcellular localization of ErbB2, specific glycan-recognizing monoclonal antibodies and lectins were used for the immunofluorescent detection of the glycan structures expressed at the cell membrane of NCI-N87 cells (Figure 3). The obtained results unveiled a complex glycosylation signature, encompassing a range of structural and functionally diverse glycan structures, many of them previously described as key molecular players in the onset and progression of human cancer [12]. Figure 2. Real-time quantitative PCR (RT-qPCR) analysis of the relative transcript expression levels of eight cancer-relevant glycosyltransferase-coding genes (FUT3,FUT8,MGAT3,MGAT5,ST3GAL3, ST3GAL4,ST6GAL1, and B3GALT5) in four GC cell lines (NCI-N87, AGS, MKN45 and MKN74). For each cell line, the mRNA levels of a given gene are presented as 2 −∆∆Ct . Target gene relative abundance was normalized to the mRNA levels of the RNA18S5 endogenous control. The obtained RT-qPCR uncovered a distinct glycosyltransferase expression signature for each of the screened GC cell lines. In particular, the B3GALT5 gene showed an expression range of multiple orders of magnitude across the four screened cell lines. Indeed, the diffuse type MKN45 cell line exhibits remarkably higher levels of this enzyme’s mRNA when compared to the other cell lines, especially in the case of intestinal type MKN74 cells where no mRNA was detected. A similar expression pattern was observed for ST6GAL1. However, regarding ST6GAL1, a generally higher transcript expression was consistently observed across the four screened GC cell lines, with the exception of MKN74, in comparison to the remaining screened glyco-genes. Illustrative of the cell line-specific glycosyltransferase expression signature, the ErbB2-overexpressing NCI-N87 cell line displayed the highest expression of FUT3,MGAT5 and ST3GAL4 genes, but also the lowest detected transcript levels of both FUT8 and ST3GAL3. The obtained results are in accordance with previous data published by our group, in respect to the GC cell lines and glycosyltransferase-coding genes commonly analyzed in both studies [23]. 2.3. Glycosylation Profile of ErbB2-Overexpressing NCI-N87 Cells Since the NCI-N87 GC cell line exhibited the highest levels of both ErbB2 expression and activation, it was therefore selected for further glycan epitope characterization. Due to the subcellular localization of ErbB2, specific glycan-recognizing monoclonal antibodies and lectins were used for the immunofluorescent detection of the glycan structures expressed at the cell membrane of NCI-N87 cells Int. J. Mol. Sci. 2017,18, 2262 5 of 19 (Figure 3). The obtained results unveiled a complex glycosylation signature, encompassing a range of structural and functionally diverse glycan structures, many of them previously described as key molecular players in the onset and progression of human cancer [12]. Int. J. Mol. Sci. 2017, 18, 2262 5 of 18 Figure 3. Immunofluorescent labeling of the glycan epitopes expressed at the cell membrane of the NCI-N87 cell line. The specifications of the used glycan-recognizing monoclonal antibodies (MAbs) (Lewis a (Lea), Lewis x (Lex), Lewis b (Leb), Lewis y (Ley), sialyl Lewis a (SLea), sialyl Lewis x (SLex) and sialyl Tn (STn)) and lectins (Sambucus nigra (SNA), Aleuria aurantia (AAL), Concanavalin A (ConA), Phaseolus vulgaris erythroagglutinin (PHA-E) and Phaseolus vulgaris leucoagglutinin (PHAL)) are listed in Table 1. White scale bar indicates 50 μm. All experiments were performed in duplicate and a representative image for each antigen is depicted. The cell membrane of NCI-N87 cells stained positive for all neutral (Lea, Leb and Lewis y (Ley)) and sialylated (SLea and sialyl Lewis x (SLex)) forms of the Lewis antigens, with the exception of Lewis x (Lex), for which no signal was detected. The expression of the tumor-associated sialyl Tn (STn) truncated O-glycan antigen was also undetected. This observation suggests that the detected terminal α2,6 sialic acid moieties by the lectin SNA are present on N-glycan chains. The presence at the cellular membrane of four additional cancer-relevant glycan structures (terminal and core fucose, high mannose, and bisecting and branching GlcNAc) was detected through their specific recognition by a panel of carbohydrate-binding lectins (Aleuria aurantia (AAL), Concanavalin A (ConA), Phaseolus vulgaris erythroagglutinin (PHA-E) and Phaseolus vulgaris leucoagglutinin (PHA-L), respectively). Regarding the enzymes responsible for the biosynthesis of distinct glycan determinants, these results are in agreement with the previously characterized transcription profile of this particular cell line (Figure 2). For example, the presence of SLex, terminal α2,6 sialic acid, bisecting and branched complex N-glycan determinants, consistently match the detected expression of the ST3GAL4, ST6GAL1, MGAT3 and MGAT5 genes, respectively. 2.4. ErbB2 Glycan Signatures in NCI-N87 Cells With the aim of elucidating the ErbB2 glycosylation pattern in NCI-N87 cells, the receptor was immunoprecipitated from NCI-N87 total cell lysates. The yield and purity of receptor immunoprecipitation was evaluated by silver staining, and ErbB2 was validated by the subsequent peptide mass fingerprint analysis of the major 185-kDa band by matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF-TOF) mass spectrometry (Figure 4a). The visible bands at 50 kDa correspond to the IgG heavy chains of the ErbB2-specific MAb used for receptor immunoprecipitation. As a control, a parallel immunoprecipitation reaction using normal rabbit IgGs was performed. As no signal in ErbB2 WB was detected, the specificity of receptor’s immunoprecipitation was validated (Figure 4b). Figure 3. Immunofluorescent labeling of the glycan epitopes expressed at the cell membrane of the NCI-N87 cell line. The specifications of the used glycan-recognizing monoclonal antibodies (MAbs) (Lewis a (Le a ), Lewis x (Le x ), Lewis b (Le b ), Lewis y (Le y ), sialyl Lewis a (SLe a ), sialyl Lewis x (SLe x ) and sialyl Tn (STn)) and lectins (Sambucus nigra (SNA), Aleuria aurantia (AAL), Concanavalin A (ConA), Phaseolus vulgaris erythroagglutinin (PHA-E) and Phaseolus vulgaris leucoagglutinin (PHA-L)) are listed in Table 1. White scale bar indicates 50 µ m. All experiments were performed in duplicate and a representative image for each antigen is depicted. The cell membrane of NCI-N87 cells stained positive for all neutral (Le a , Le b and Lewis y (Le y )) and sialylated (SLe a and sialyl Lewis x (SLe x )) forms of the Lewis antigens, with the exception of Lewis x (Le x ), for which no signal was detected. The expression of the tumor-associated sialyl Tn (STn) truncated O-glycan antigen was also undetected. This observation suggests that the detected terminal α 2,6 sialic acid moieties by the lectin SNA are present on N-glycan chains. The presence at the cellular membrane of four additional cancer-relevant glycan structures (terminal and core fucose, high mannose, and bisecting and branching GlcNAc) was detected through their specific recognition by a panel of carbohydrate-binding lectins (Aleuria aurantia (AAL), Concanavalin A (ConA), Phaseolus vulgaris erythroagglutinin (PHA-E) and Phaseolus vulgaris leucoagglutinin (PHA-L), respectively). Regarding the enzymes responsible for the biosynthesis of distinct glycan determinants, these results are in agreement with the previously characterized transcription profile of this particular cell line (Figure 2). For example, the presence of SLe x , terminal α 2,6 sialic acid, bisecting and branched complex N-glycan determinants, consistently match the detected expression of the ST3GAL4,ST6GAL1,MGAT3 and MGAT5 genes, respectively. 2.4. ErbB2 Glycan Signatures in NCI-N87 Cells With the aim of elucidating the ErbB2 glycosylation pattern in NCI-N87 cells, the receptor was immunoprecipitated from NCI-N87 total cell lysates. The yield and purity of receptor immunoprecipitation was evaluated by silver staining, and ErbB2 was validated by the subsequent peptide mass fingerprint analysis of the major 185-kDa band by matrix-assisted laser desorption Int. J. Mol. Sci. 2017,18, 2262 6 of 19 ionization time-of-flight (MALDI/TOF-TOF) mass spectrometry (Figure 4a). The visible bands at 50 kDa correspond to the IgG heavy chains of the ErbB2-specific MAb used for receptor immunoprecipitation. As a control, a parallel immunoprecipitation reaction using normal rabbit IgGs was performed. As no signal in ErbB2 WB was detected, the specificity of receptor’s immunoprecipitation was validated (Figure 4b). For the profiling of the glycan structures specifically carried by ErbB2 in NCI-N87 cells, the immunoprecipitated receptor was further characterized by both WB and lectin blot analysis with the previously used panel of glycan-recognizing antibodies and lectins found positive by immunofluorescence in NCI-N87 cells (Figure 4c). The 185-kDa band identified as the ErbB2 receptor displayed a specific glycosylation profile resembling the one of NCI-N87 cells. The only exception was SLe x , for which no signal was observed by WB. Indeed, when SLe x WB was performed on NCI-N87 whole cell lysates, a single band was visible with a corresponding molecular weight higher than 185-kDa, indicating that the SLe x epitope is carried by a protein other than ErbB2 in this particular GC cell line (data not shown). To our knowledge, this is the first study identifying ErbB2 as a carrier of the tumor-associated SLea, a well-established molecular player in the malignant behavior of tumor cells. Int. J. Mol. Sci. 2017, 18, 2262 6 of 18 For the profiling of the glycan structures specifically carried by ErbB2 in NCI-N87 cells, the immunoprecipitated receptor was further characterized by both WB and lectin blot analysis with the previously used panel of glycan-recognizing antibodies and lectins found positive by immunofluorescence in NCI-N87 cells (Figure 4c). The 185-kDa band identified as the ErbB2 receptor displayed a specific glycosylation profile resembling the one of NCI-N87 cells. The only exception was SLex, for which no signal was observed by WB. Indeed, when SLex WB was performed on NCIN87 whole cell lysates, a single band was visible with a corresponding molecular weight higher than 185-kDa, indicating that the SLex epitope is carried by a protein other than ErbB2 in this particular GC cell line (data not shown). To our knowledge, this is the first study identifying ErbB2 as a carrier of the tumor-associated SLea, a well-established molecular player in the malignant behavior of tumor cells. Figure 4. Glycan profiling of ErbB2 immunoprecipitated from NCI-N87 cells. (a) Silver staining of immunoprecipitated ErbB2 from NCI-N87 whole cell lysates and confirmation of receptor identity by matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF-TOF) mass spectrometry. The excised and identified 185-kDa band is marked in red. The summary of the Peptide Mass Fingerprint analysis is also depicted; (b) ErbB2 WB for validation of the immunoprecipitation reaction. Normal rabbit IgGs were used as a control of the immunoprecipitation reaction; (c) WB and lectin blot analysis of ErbB2 glycan structures using glycan-recognizing MAbs and lectins (specifications are listed in Table 1). All WB experiments were performed in duplicate and a single representative analysis for each antigen and lectin is depicted. 2.5. Interplay Between ErbB2 and Sialyl Lewis a (SLea) Expression Having identified ErbB2 as a protein carrier of the SLea antigen in ErbB2-overexpressing NCIN87 cells, we aimed at dissecting the biosynthetic pathway of this cancer-relevant glycan epitope in this particular cell line, both in vitro and in silico. Lewis antigens constitute terminal structures in both Nand O-linked oligosaccharide chains [24]. To elucidate which type of SLea-capped glycans are carried by ErbB2 in NCI-N87 cells, the immunoprecipitated receptor was digested with the PNGase F enzyme, a glycosidase capable of removing all N-linked glycans through the specific cleavage between the sugar’s innermost GlcNAc and the protein’s asparagine residue (Figure 5a). As expected, following PNGase F digestion, a clear shift in the molecular weight of ErbB2 was observed. This shift was accompanied by the disappearance of the 185-kDa SLea band, indicating that this terminal carbohydrate epitope is present on N-linked glycan chains. Figure 4. Glycan profiling of ErbB2 immunoprecipitated from NCI-N87 cells. ( a ) Silver staining of immunoprecipitated ErbB2 from NCI-N87 whole cell lysates and confirmation of receptor identity by matrix-assisted laser desorption ionization time-of-flight (MALDI/TOF-TOF) mass spectrometry. The excised and identified 185-kDa band is marked in red. The summary of the Peptide Mass Fingerprint analysis is also depicted; ( b ) ErbB2 WB for validation of the immunoprecipitation reaction. Normal rabbit IgGs were used as a control of the immunoprecipitation reaction; ( c ) WB and lectin blot analysis of ErbB2 glycan structures using glycan-recognizing MAbs and lectins (specifications are listed in Table 1). All WB experiments were performed in duplicate and a single representative analysis for each antigen and lectin is depicted. 2.5. Interplay Between ErbB2 and Sialyl Lewis a (SLea) Expression Having identified ErbB2 as a protein carrier of the SLe a antigen in ErbB2-overexpressing NCI-N87 cells, we aimed at dissecting the biosynthetic pathway of this cancer-relevant glycan epitope in this particular cell line, both in vitro and in silico. Lewis antigens constitute terminal structures in both Nand O-linked oligosaccharide chains [ 24 ]. To elucidate which type of SLe a -capped glycans are carried by ErbB2 in NCI-N87 cells, the immunoprecipitated receptor was digested with the PNGase F enzyme, a glycosidase capable of removing all N-linked glycans through the specific cleavage between the sugar’s innermost GlcNAc and the protein’s asparagine residue (Figure 5a). As expected, following PNGase F digestion, a clear shift in the molecular weight of ErbB2 was observed. This shift was accompanied by the disappearance of the 185-kDa SLe a band, indicating that this terminal carbohydrate epitope is present on N-linked glycan chains. Int. J. Mol. Sci. 2017,18, 2262 7 of 19 Int. J. Mol. Sci. 2017, 18, 2262 7 of 18 Figure 5. Association between ErbB2 expression and biosynthesis of N-linked SLea in GC cell lines. (a) WB of ErbB2 and SLea following receptor immunoprecipitation from NCI-N87 whole cell lysates and PNGase F digestion; (b) Left panel: Schematic representation of the glycosyltransferases (1–10) and enzymes responsible for the assembly of activated monosaccharides (A–I) integrating the biosynthetic pathways of Lewis antigens; Right panel: Relative transcriptomic abundance of the genes represented in the right panel across the four screened GC cell lines. The raw data on transcription levels were extracted from the Barretina CellLine data deposited in the OncomineTM database. Based on normalized transcription values of all 35 GC cell lines that were included in the dataset, a range of average expression values for GC cell lines was defined for each gene. Transcription values that were exceptionally high are highlighted in green and values that were exceptionally low in red; (c) WB of SLea expression in four GC cell lines. All experiments were performed in duplicate and a representative WB analysis is depicted; (d) Spearman’s rank correlation between the transcript levels of the ERBB2 and FUT3 genes in GC patients from the Ooi dataset available at the OncomineTM database. Figure 5. Association between ErbB2 expression and biosynthesis of N-linked SLe a in GC cell lines. ( a ) WB of ErbB2 and SLe a following receptor immunoprecipitation from NCI-N87 whole cell lysates and PNGase F digestion; ( b ) Left panel: Schematic representation of the glycosyltransferases (1–10) and enzymes responsible for the assembly of activated monosaccharides (A–I) integrating the biosynthetic pathways of Lewis antigens; Right panel: Relative transcriptomic abundance of the genes represented in the right panel across the four screened GC cell lines. The raw data on transcription levels were extracted from the Barretina CellLine data deposited in the Oncomine TM database. Based on normalized transcription values of all 35 GC cell lines that were included in the dataset, a range of average expression values for GC cell lines was defined for each gene. Transcription values that were exceptionally high are highlighted in green and values that were exceptionally low in red; ( c ) WB of SLe a expression in four GC cell lines. All experiments were performed in duplicate and a representative WB analysis is depicted; ( d ) Spearman’s rank correlation between the transcript levels of the ERBB2 and FUT3 genes in GC patients from the Ooi dataset available at the OncomineTM database. Int. J. Mol. Sci. 2017,18, 2262 8 of 19 To establish a valid causal association between the performed transcriptomic analysis of glycosyltransferase-coding genes and the expression of SLe a in the NCI-N87 GC cell line (Figures 2 and 3, respectively), in silico analysis, using the transcriptomic Barretina CellLine data deposited in the Oncomine TM platform (www.oncomine.org), was performed. For this purpose, the either exceptionally high or low expression of each gene of interest, as compared to a total of 35 GC cell lines, was assessed in the four GC cell lines (Figure 5b, right panel). Amongst the four analyzed GC cell lines, NCI-N87 clearly exhibited the most robust expression profile of genes relevant for SLe a biosynthesis and thus emerged as the most promising candidate to consistently express this glycan epitope. Besides the pronounced overexpression of SLe a -relevant genes (FUT3,TSTA3,SLC35C1, and SLC35A1), no downregulation of enzyme-coding genes directly implicated in SLe a biosynthesis is observed, contrary to what occurs for the remaining three GC cell lines. The generated in silico results are in robust alignment with the ones obtained in the performed transcriptomic analysis (Figure 2). Illustrating this observation is the overexpression of FUT3 by the ERBB2-amplified NCI-N87, and of B3GALT5 by MKN45. It is also worth noting that, although unrelated to SLe a biosynthesis, ST3GAL4 is downregulated in AGS cells, and ST6GAL1 is downregulated in MKN45, NCI-N87 and AGS when compared to MKN74 cell line. To validate the robustness of the in silico data, the four GC cell lines were screened for the expression of SLe a by WB (Figure 5c). Remarkably, and in agreement with the in silico results, the only GC cell line exhibiting a strong SLe a immunodetection was NCI-N87. Our data therefore suggest that the marked upregulation of SLe a in NCI-N87 cells may be driven by high FUT3 expression levels. Finally, we sought to investigate whether the apparent ErbB2-SLe a crosstalk could also be observed in patients harboring GC. For this purpose, an in silico analysis based on the transcriptomic data of the Ooi dataset extracted from the Oncomine TM platform was carried out. Among the 200 primary gastric carcinomas of the referred dataset, a statistically highly significant positive association (Spearman’s rank correlation p< 0.0001) between the expression of ERBB2 and FUT3 genes was observed (Figure 5d). The latter gene encodes for the FucT-III glycosyltransferase, also known as the Lewis enzyme, which is solely responsible for the biosynthesis of SLe a [ 25 ]. In conclusion, a higher ERBB2 expression was associated to FUT3 upregulation in the GC patients, and vice-versa. 2.6. Inhibition of Glycosylation and Glycan Epitope-Blocking Disrupt ErbB2 Expression and Activation Protein N-linked glycosylation actively regulates RTK maturation, translocation to the cell membrane and signaling activity [ 26 ]. Having identified the major types of glycans carried by ErbB2 in NCI-N87 cells, we aimed at assessing the functional impact of glycosylation-targeting agents on receptor’s expression and activation status in this particular cell line. NCI-N87 cells were subjected to increasing concentrations of two distinct treatment regimens: tunicamycin, a natural inhibitor of the enzyme catalyzing the first step of N-glycan biosynthesis, and the CA 19.9 MAb targeting the SLe a epitope. Following 24 h of treatment, WB analysis of both total and phosphorylated forms of ErbB2 was performed on freshly collected protein extracts (Figure 6). Only the highest concentration of the tunicamycin compound (1000 ng/mL) exerted a pronounced effect on the levels of both total and activated protein (Figure 6a). In regard to total receptor’s expression, a second band with lower molecular weight strongly indicates the presence of a deglycosylated version of ErbB2, in addition to the heavier 185-kDa band corresponding to the fully glycosylated receptor. Moreover, the same concentration of tunicamycin led to a significant reduction of receptor’s phosphorylation. Since ErbB2 phosphorylation seems to be only detected at 185-kDa, the observed reduction can be due to a general decrease in the abundance of the fully matured transmembrane receptor, which is the one targeted by phosphorylation. As for the treatment of NCI-N87 cells with the CA 19.9 MAb, drastic effects on both forms of the receptor could also be clearly observed. While the lowest CA 19.9 concentration (0.2 µ g/mL) produced no visible effects on ErbB2 total expression, and a mild, yet detectable, reduction on the ErbB2 phosphorylation, the highest MAb concentration (2 µ g/mL) led to a severe reduction of total receptor’s levels and a complete abrogation of receptor’s phosphorylation. Int. J. Mol. Sci. 2017,18, 2262 9 of 19 Int. J. Mol. Sci. 2017, 18, 2262 9 of 18 Figure 6. WB analysis of the total and phosphorylated ErbB2 following in vitro deglycosylation or MAb-mediated blockage of the SLea epitope in the NCI-N87 cell line. Cells were treated for 24 h with different concentrations of: (a) tunicamycin in complete growth medium; and (b) CA 19.9 MAb diluted in simple growth medium with the corresponding controls. α-Tubulin was used as a loading control. Band densities, normalized for the loading control (for total ErbB2) or both the loading control and total receptor (for pErbB2), are depicted. All experiments were performed in duplicate and a representative WB analysis is depicted. CGM: complete growth medium. 3. Discussion The extracellular region of the ErbB2 mitogenic receptor is a well-documented target for extensive glycosylation, which has been shown to actively promote human carcinogenesis through the regulation of receptor’s total expression, subcellular localization and signaling potential [15,26,27]. However, the ErbB2 glycosylation signature in GC cells and the molecular mechanisms through which it may tune receptor biology towards malignancy remain poorly understood. The present study aimed at disclosing glycan determinants carried by ErbB2 that may act as functional regulators of the receptor in ErbB2-driven gastric carcinogenesis. The NCI-N87 cell line was therefore selected, amongst the four screened cell lines, as the most appropriate in vitro model of ErbB2positive GC. Despite the similar high levels of total EGFR expression observed in NCI-N87 and MKN74, the former GC cell line exhibited the most pronounced activation of this receptor, and also of ErbB2, as previously described [28]. This observation may reflect the homoor hetero-oligomerization capacity of the ErbB transmembrane receptors, which constitutes a well-established pre-requisite for intracellular activation [29]. The ErbB2 protein is overexpressed in NCI-N87 cells as a direct consequence of gene amplification and constitutes the preferred heterodimerization partner of EGFR [30,31]. Therefore, in the NCI-N87 GC cell line, the high levels of expression of the ErbB2 RTK at the cell membrane lead not only to its own hyperactivation, but also of the remaining members of the family. This is in agreement with the significantly lower levels of both EGFR endogenous activation ErbB2 total expression observed in the three other GC cell lines. However, whether ErbB2-specific glycosylation plays an active part on dimer formation, although reported for other ErbB receptors, remains a question to be addressed [16,32–35]. The elaborate shaping of the cellular glycan landscape depends on the highly controlled and dynamic crosstalk between both glycosyltransferases and glycosidases [36]. Interestingly, the observed expression levels of the selected glycogenes agree with the known competing enzymatic role that these glycosyltransferases play in the cell, in response to both external and internal stimuli. For Figure 6. WB analysis of the total and phosphorylated ErbB2 following in vitro deglycosylation or MAb-mediated blockage of the SLe a epitope in the NCI-N87 cell line. Cells were treated for 24 h with different concentrations of: ( a ) tunicamycin in complete growth medium; and ( b ) CA 19.9 MAb diluted in simple growth medium with the corresponding controls. α -Tubulin was used as a loading control. Band densities, normalized for the loading control (for total ErbB2) or both the loading control and total receptor (for pErbB2), are depicted. All experiments were performed in duplicate and a representative WB analysis is depicted. CGM: complete growth medium. 3. Discussion The extracellular region of the ErbB2 mitogenic receptor is a well-documented target for extensive glycosylation, which has been shown to actively promote human carcinogenesis through the regulation of receptor’s total expression, subcellular localization and signaling potential [ 15 , 26 , 27 ]. However, the ErbB2 glycosylation signature in GC cells and the molecular mechanisms through which it may tune receptor biology towards malignancy remain poorly understood. The present study aimed at disclosing glycan determinants carried by ErbB2 that may act as functional regulators of the receptor in ErbB2-driven gastric carcinogenesis. The NCI-N87 cell line was therefore selected, amongst the four screened cell lines, as the most appropriate in vitro model of ErbB2-positive GC. Despite the similar high levels of total EGFR expression observed in NCI-N87 and MKN74, the former GC cell line exhibited the most pronounced activation of this receptor, and also of ErbB2, as previously described [ 28 ]. This observation may reflect the homoor hetero-oligomerization capacity of the ErbB transmembrane receptors, which constitutes a well-established pre-requisite for intracellular activation [ 29 ]. The ErbB2 protein is overexpressed in NCI-N87 cells as a direct consequence of gene amplification and constitutes the preferred heterodimerization partner of EGFR [ 30 , 31 ]. Therefore, in the NCI-N87 GC cell line, the high levels of expression of the ErbB2 RTK at the cell membrane lead not only to its own hyperactivation, but also of the remaining members of the family. This is in agreement with the significantly lower levels of both EGFR endogenous activation ErbB2 total expression observed in the three other GC cell lines. However, whether ErbB2-specific glycosylation plays an active part on dimer formation, although reported for other ErbB receptors, remains a question to be addressed [16,32–35]. The elaborate shaping of the cellular glycan landscape depends on the highly controlled and dynamic crosstalk between both glycosyltransferases and glycosidases [ 36 ]. Interestingly, the observed expression levels of the selected glycogenes agree with the known competing enzymatic role that these glycosyltransferases play in the cell, in response to both external and internal stimuli. For example, the Int. J. Mol. Sci. 2017,18, 2262 16 of 19 10. 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