| Background Gastric cancer(GC) ranks the second most commonly diagnosed malignancy and the second most lethal cancer in China. Despite the great improvements in MDR(multi-drug resistance) study of gastric cancer, MDR remains a great obstacle to effective chemotherapy for gastric cancer. The development of MDR in tumor is a complex biological process involving multiple molecules and signaling pathways. However, previous studies mainly focused on the role of specific protein expression, particularly intracellular protein expression, in tumor MDR, but provided little information about protein glycosylation.As one of the most abundant and important post-translational modifications(PTMs), glycosylation exercises great influences on not only structures but also functions of glycoproteins. Protein glycosylation is critical for tumor development, proliferation, invasion and metastasis, etc. Besides, glycomic alterations of glycoproteins exerted enormous effects on chemosensitivity of cancers. Several studies indicated that glycosylation of specific protein played a major role in the development of MDR in tumor, and specific glycoform variations could regulate the efficacy of chemotherapy. In recent years, with our increasingly understanding the role of tumor microenvironment in tumor initiation and development, the contribution of secreted proteins of tumor cells to MDR has been widely recognized. A number of secreted glycoproteins have been reported to be involved in the development of MDR in gastric cancer. However, whether glycosylation of secreted glycoproteins changes during MDR of gastric cancer is unclear. Based on our previous studies on tumor cell protein glycosylation, our present work fabricated a platform for the identification and quantification of site-specific glycosylation of secreted proteins in our MDR cell model, and further identified several MDR-associated aberrantly glycosylated secreted glycoproteins, thus providing new insights into the sophisticated mechanisms underlying MDR in gastric cancer.Objectives1. To establish a comprehensive analyzing method for characterizing the site-specific glycoforms of secreted glycoproteins from gastric cancer cells;2. To identify the glycosylation alterations of secreted proteins from gastric cancer cells during MDR through high-throughput MS analysis;3. To identify several promising MDR-associated secreted proteins from the point of glycosylation, thus facilitating our further study on MDR in GC.Methods1. The serum-free culture medium was collected and then concentrated by ultra filters. Then, the concentrated secreted proteins were digested by trypsin. Additionally, the glycopeptides were enriched by using hydrophilic interaction liquid chromatography(HILIC), which could retain the glycan structures linked to glycosites with high specificity.2. By MS analysis, we could get the raw data of de-glycopeptides and intact glycopeptides, respectively.3. The determination of site-specific glycoforms was performed by using Ar Mone proteomics data processing platform; and the Perseus software was employed for the quantification analysis of site-specific glycoforms.4. We further validated the possible correlations between AXL and MDR in GC through analyzing TCGA GC data and western blot experiments.Results1. Generation of secretome datasets from 3 gastric cancer cell linesGenerally speaking, a total of 1033 N-linked glycosites(localization probability > 0.75), mapping to 436 non-redundant N-glycoproteins, were quantified from secreted proteins of the three cell lines(SGC7901, SGC7901/ADR and SGC7901/VCR). In addition, we totally identified 2222 N-linked site-specific glycoforms with high confidence. To verify whether the identified glycoproteins were secretory proteins, we applied bioinformatics softwares and Uniprot database to predict protein secretion pathway. 272 proteins were predicted to be secreted in the classical secretory pathway; 74 proteins were secreted through the nonclassical secretory pathway, demonstrating high efficiency of the platform for analysis of secreted protein glycosylation.2. Characterization of N-glycosite occupancy varying with MDRTotally, 240 N-glycosites were found dramatically changed in SGC7901-ADR/VCR(124 increased and 116 decreased) compared to SGC7901. And the significantly different glycosites could be mapped to 163 glycoproteins(84 glycoproteins for up-regulated glycosites, 76 glycoproteins for down-regulated glycosites). Interestingly, there are 3 proteins(IGF2R, ITGB1 and PTPRF) that both have up-regulated and down-regulated glycosites. Further bioinformatics analysis indicated that the leading three molecular functions of 87 glycoproteins for increased glycosites were binding(32.4%), receptor activity(25.4%) and catalytic activity(23.9%), while for 79 glycoproteins corresponding to decreased glycosites, catalytic activity(33.3%), receptor activity(25.0%) and binding(25.0%) ranked the top three.3. Identification of altered site-specific glycoforms between SGC7901 and its MDR counterpartsIn the present study, we merely found increased core-fucosylation and hybrid glycans after acquired drug resistance in gastric cancer cell lines, while the other major glycoforms did not show distinct changes. Moreover, we further characterized the significantly different site-specific glycoforms between SGC7901 and its MDR counterparts. 175 site-specific glycoforms were increased in secreted proteins of MDR cell lines, while 324 glycoforms were decreased. Interestingly, we discovered that some glycosites contained both upregulated and down-regulated glycoforms, indicating the high complexity of glycosylation. Totally, 499 site-specific glycoforms onto 151 glycosites in 106 glycoproteins were significantly changed between SGC7901 and its MDR derivates.4. Integrative characterization of changed glycosites and altered site-specific glycoformsNotably, we found that the glycosites with significant changes on glycosite-level rarely intersected with those corresponding to altered site-specific glycoforms. And a total number of 57 glycoproteins with more than 2 significantly different glycoforms onto one specific glycosite were identified. 33 of these glycoproteins had been reported to be associated with MDR of multiple cancers by searching in Pub Med.5. Preliminary validation of AXL's role in GC MDROur further work validated the increased m RNA expression of AXL in GC MDR cells and increased protein expression of soluble AXL in the conditioned medium of GC MDR cells. In addition, we found that high expression of tumor AXL predicted worse survival in GC patients based on TCGA database. In a word, glycosylation and expression differences of soluble AXL between GC drug-sensitive and resistant cells have been observed, suggesting a significant role of soluble AXL in the MDR of GC.ConclusionIn the current study, we provided the comprehensive secretome glycoproteomic characterization of SGC7901 and its MDR counterparts SGC7901/ADR, SGC7901/VCR. And, we manifested that N-glycosites and site-specific glycoforms of secreted proteins in drug-resistant cell lines were distinctly different from those in the parental cell line for the first time. Further characterization highlighted the significance of some aberrantly glycosylated secretory proteins in MDR, suggesting that manipulating the glycosylation of specific glycoproteins could be a potential target for overcoming multi-drug resistance in gastric cancer. |
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