Bisecting GlcNAc modification reverses the chemoresistance via attenuating the function of P-gp.

Rationale: Chemoresistance is a key factor contributing to the failure of anti-breast cancer chemotherapy. Although abnormal glycosylation is closely correlated with breast cancer progression, the function of glycoconjugates in chemoresistance remains poorly understood. Methods: Levels and regulatory roles of bisecting N-acetylglucosamine (GlcNAc) in chemoresistant breast cancer cells were determined in vitro and in vivo. Glycoproteomics guided identification of site-specific bisecting GlcNAc on P-glycoprotein (P-gp). Co-immunoprecipitation coupled mass spectrometry (Co-IP-MS) and proximity labelling MS identified the interactome of P-gp, and the biological function of site-specific bisecting GlcNAc was investigated by site/truncation mutation and structural simulations. Results: Bisecting GlcNAc levels were reduced in chemoresistant breast cancer cells, accompanied by an enhanced expression of P-gp. Enhanced bisecting GlcNAc effectively reversed chemoresistance. Mechanical study revealed that bisecting GlcNAc impaired the association between Ezrin and P-gp, leading to a decreased expression of membrane P-gp. Bisecting GlcNAc suppressed VPS4A-mediated P-gp recruitment into microvesicles, and chemoresistance transmission. Structural dynamics analysis suggested that bisecting GlcNAc at Asn494 introduced structural constraints that rigidified the conformation and suppressed the activity of P-gp. Conclusion: Our findings highlight the crucial role of bisecting GlcNAc in chemoresistance and suggest the possibility of reversing chemoresistance by modulating the specific glycosylation in breast cancer therapy.

Integrated pan-cancer analysis and experimental verification of the roles of meiotic nuclear divisions 1 in breast cancer.

INTRODUCTION: The aberrant up-regulation of meiotic nuclear division 1 (MND1) in somatic cells is considered as one of the driving factors of oncogenesis, whereas its expression and role in breast invasive cancer (BRCA) remain unclear. Hence, this study embarked on a comprehensive evaluation of MND1 across various cancers and identified its roles in BRCA. METHODS: Based on publicly available databases, including but not limited to UCSC Xena, TCGA, GTEx, GEO, STRING, GeneMANIA, and CancerSEA, we evaluated the expression patterns, genomic features, and biological functions of MND1 from a pan-cancer viewpoint and delved into the implications of MND1 in the prognosis and treatment of BRCA. Further molecular biology experiments were undertaken to identify the role of MND1 in proliferation, migration, and apoptosis in BRCA cells. RESULTS: Elevated levels of MND1 were notably observed in a wide array of tumor types, especially in BRCA, COAD, HNSC, LIHC, LUAD, LUSC, STAD, and UCEC. Elevated MND1 expression was markedly associated with shortened OS in several tumors, including BRCA (HR = 1.52 [95%CI, 1.10-2.09], P = 0.011). The up-regulation of MND1 in BRCA was validated in external cohorts and clinical samples. Survival analyses demonstrated that elevated MND1 expression was associated with decreased survival for patients with BRCA. Co-expressed genes of MND1 were identified, and subsequent pathway analyses based on significantly associated genes indicated that MND1 plays key roles in DNA replication, cell cycle regulation, and DNA damage repair. The observed abnormal elevation and activation of MND1 led to increased proliferation and migration, along with decreased apoptosis in BRCA cells. CONCLUSIONS: MND1 emerges as a promising biomarker for diagnostic and therapeutic targeting in various cancers, including BRCA. The abnormal up-regulation and activation of MND1 are linked to carcinogenesis and poor prognosis among BRCA patients, which may be attributed to its involvement in HR-dependent ALT, warranting further scrutiny.

The trajectory of vesicular proteomic signatures from HBV-HCC by chitosan-magnetic bead-based separation and DIA-proteomic analysis.

Hepatocellular carcinoma (HCC) is a prevalent primary liver cancer often associated with chronic hepatitis B virus infection (CHB) and liver cirrhosis (LC), underscoring the critical need for biomarker discovery to improve patient outcomes. Emerging as a promising avenue for biomarker development, proteomic technology leveraging liquid biopsy from small extracellular vesicles (sEV) offers new insights. Here, we evaluated various methods for sEV isolation and identified polysaccharide chitosan (CS) as an optimal approach. Subsequently, we employed optimized CS-based magnetic beads (Mag-CS) for sEV separation from serum samples of healthy controls, CHB, LC, and HBV-HCC patients. Leveraging data-independent acquisition mass spectrometry coupled with machine learning, we uncovered potential vesicular protein biomarker signatures (KNG1, F11, KLKB1, CAPNS1, CDH1, CPN2, NME2) capable of distinguishing HBV-HCC from CHB, LC, and non-HCC conditions. Collectively, our findings highlight the utility of Mag-CS-based sEV isolation for identifying early detection biomarkers in HBV-HCC.

Integrating transcriptomics, glycomics and glycoproteomics to characterize hepatitis B virus-associated hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) ranks as the third most common cause of cancer related death globally, representing a substantial challenge to global healthcare systems. In China, the primary risk factor for HCC is the hepatitis B virus (HBV). Aberrant serum glycoconjugate levels have long been linked to the progression of HBV-associated HCC (HBV-HCC). Nevertheless, few study systematically explored the dysregulation of glycoconjugates in the progression of HBV-associated HCC and their potency as the diagnostic and prognostic biomarker. METHODS: An integrated strategy that combined transcriptomics, glycomics, and glycoproteomics was employed to comprehensively investigate the dynamic alterations in glyco-genes, N-glycans, and glycoproteins in the progression of HBV- HCC. RESULTS: Bioinformatic analysis of Gene Expression Omnibus (GEO) datasets uncovered dysregulation of fucosyltransferases (FUTs) in liver tissues from HCC patients compared to adjacent tissues. Glycomic analysis indicated an elevated level of fucosylated N-glycans, especially a progressive increase in fucosylation levels on IgA1 and IgG2 determined by glycoproteomic analysis. CONCLUSIONS: The findings indicate that the abnormal fucosylation plays a pivotal role in the progression of HBV-HCC. Systematic and integrative multi-omic analysis is anticipated to facilitate the discovery of aberrant glycoconjugates in tumor progression.

Sialylation on vesicular integrin ß1 determined endocytic entry of small extracellular vesicles into recipient cells.

BACKGROUND: Small extracellular vesicles (sEV) are closely associated with the development and metastasis of many types of mammalian cancer. Glycoconjugates are highly expressed on sEV and play important roles in sEV biogenesis and their interaction with other cells. However, the study on vesicular glycoconjugates are far behind proteins and nucleic acids. Especially, the functions of sialic acids which are the terminal components of glycoconjugates, are poorly understood in sEV. METHODS: Sialic acid levels on sEV from plasma and bladder cancer cells were determined by ELISA and lectin blotting. Effects of sialylation on sEV uptake were determined by flow cytometry. Vesicular glycoproteins bearing sialic acids responsible for sEV uptake was identified by proteomics and density gradient centrifugation, and their site-specific sialylation functions were assayed by N-glycosylation site mutation. Effects of integrin ß1 bearing sialic acids on the pro-metastatic function of sEV in vivo were explored using Balb/c nu/nu mice. RESULTS: (1) Increased sialic acid levels were observed in sEV from malignant bladder cancer cells. (2) Elimination of sialic acids on sEV impaired sEV uptake by recipient cells. (3) Vesicular integrin ß1 bearing sialic acids was identified to play a key role in sEV uptake. (4) Desialylation of the hybrid domain of vesicular integrin ß1 inhibited its binding to matrix fibronectin, and reduced sEV entry into recipient cells. (5) Sialylation on integrin ß1 affected pro-metastatic function of sEV in Balb/c nu/nu mice. CONCLUSIONS: Taken together, our findings indicate important functional roles of sialic acids in sEV uptake and reprogramming plasticity of surrounding normal epithelial cells.

Optimizing and integrating depletion and precipitation methods for plasma proteomics through data-independent acquisition-mass spectrometry.

The application of plasma proteomics is a reliable approach for the discovery of biomarkers. However, the utilization of mass spectrometry-based proteomics in plasma encounters limitations due to the presence of high-abundant proteins (HAPs) and the vast dynamic range. To address this issue, we conducted an optimization and integration of depletion and precipitation strategies eliminating interference from HAPs. The optimized procedure involved utilizing 40 µL of beads for the removal of 1 µL of plasma, and maintaining a ratio of 1:1:1 between plasma, urea, and trichloroacetic acid for the precipitation of 50 µL of plasma. To facilitate high-throughput processing, experimental procedures were carried out utilizing 96-well plates. The depletion method identified a total of 1510 proteins, whereas the precipitated method yielded a total of 802 proteins. The integration of these methods yielded a total of 1794 proteins, including a wide concentration range spanning over 8 orders of magnitude. Furthermore, these approaches exhibited a commendable level of reproducibility, as indicated by median coefficients of variation of 14.7 % and 21.1 % for protein intensities, respectively. The integrative method was found to be effective in precisely quantifying yeast proteins that were intentionally spiked in plasma at predetermined rations of 5, 2, 0.5, and 0.2 with a high genuine positive recovery with a range of 71 % to 91 % of all yeast proteins. The use of a complementary and finely tuned approach involving depletion and precipitation demonstrates tremendous potential in the field of discovering protein biomarkers from large-scale cohort studies.

Impaired bisecting GlcNAc reprogrammed M1 polarization of macrophage.

The functions of macrophages are governed by distinct polarization phenotypes, which can be categorized as either anti-tumor/M1 type or pro-tumor/M2 type. Glycosylation is known to play a crucial role in various cellular processes, but its influence on macrophage polarization is not well-studied. In this study, we observed a significant decrease in bisecting GlcNAc during M0-M1 polarization, and impaired bisecting GlcNAc was found to drive M0-M1 polarization. Using a glycoproteomics strategy, we identified Lgals3bp as a specific glycoprotein carrying bisecting GlcNAc. A high level of bisecting GlcNAc modification facilitated the degradation of Lgals3bp, while a low level of bisecting GlcNAc stabilized Lgals3bp. Elevated levels of Lgals3bp promoted M1 polarization through the activation of the NF-кB pathway. Conversely, the activated NF-кB pathway significantly repressed the transcription of MGAT3, leading to reduced levels of bisecting GlcNAc modification on Lgals3bp. Overall, our study highlights the impact of glycosylation on macrophage polarization and suggests the potential of engineered macrophages via glycosylated modification. Video Abstract.

Biodistribution-based Administration of cGMP-compliant Human Umbilical Cord Mesenchymal Stem Cells Affects the Therapeutic Effect of Wound Healing.

BACKGROUND: Although mesenchymal stem cells (MSCs) are used as therapeutic agents for skin injury therapy, few studies have reported the effects of dosing duration and delivery frequency on wound healing. In addition, before the clinical application of MSCs, it is important to assess whether their usage might influence tumor occurrence. METHODS: We described the metabolic patterns of subcutaneous injection of hUC-MSCs using fluorescence tracing and qPCR methods and applied them to the development of drug delivery strategies for promoting wound healing. RESULTS: (i) We developed cGMP-compliant hUC-MSC products with critical quality control points for wound healing; (ii) The products did not possess any tumorigenic or tumor-promoting/inhibiting ability in vivo; (iii) Fluorescence tracing and qPCR analyses showed that the subcutaneous application of hUC-MSCs did not result in safety-relevant biodistribution or ectopic migration; (iv) Reinjecting hUC-MSCs after significant consumption significantly improved reepithelialization and dermal regeneration. CONCLUSIONS: Our findings provided a reference for controlling the quality of MSC products used for wound healing and highlighted the importance of delivery time and frequency for designing in vivo therapeutic studies.

Loss of bisecting GlcNAcylation on MCAM of bone marrow stoma determined pro-tumoral niche in MDS/AML.

Bone marrow (BM) stroma plays key roles in supporting hematopoietic stem cell (HSC) growth. Glycosylation contributes to the interactions between HSC and surrounding microenvironment. We observed that bisecting N-acetylglucosamine (GlcNAc) structures, in BM stromal cells were significantly lower for MDS/AML patients than for healthy subjects. Malignant clonal cells delivered exosomal miR-188-5p to recipient stromal cells, where it suppressed bisecting GlcNAc by targeting MGAT3 gene. Proteomic analysis revealed reduced GlcNAc structures and enhanced expression of MCAM, a marker of BM niche. We characterized MCAM as a bisecting GlcNAc-bearing target protein, and identified Asn 56 as bisecting GlcNAc modification site on MCAM. MCAM on stromal cell surface with reduced bisecting GlcNAc bound strongly to CD13 on myeloid cells, activated responding ERK signaling, and thereby promoted myeloid cell growth. Our findings, taken together, suggest a novel mechanism whereby MDS/AML clonal cells generate a self-permissive niche by modifying glycosylation level of stromal cells.

Identification of glycogene signature as a tool to predict the clinical outcome and immunotherapy response in breast cancer.

Background: Breast cancer is one of the most important diseases in women around the world. Glycosylation modification correlates with carcinogenesis and roles of glycogenes in the clinical outcome and immune microenvironment of breast cancer are unclear. Methods: A total of 1297 breast cancer and normal cases in the TCGA and GTEx databases were enrolled and the transcriptional and survival information were extracted to identify prognostic glycogenes using Univariate Cox, LASSO regression, Multivariate Cox analyses and Kaplan-Meier method. The immune infiltration pattern was explored by the single sample gene set enrichment method. The HLA and immune checkpoint genes expression were also compared in different risk groups. The expressions of a glycogene MGAT5 as well as its products were validated by immunohistochemistry and western blotting in breast cancer tissues and cells. Results: A 19-glycogene signature was identified to separate breast cancer patients into high- and low-risk groups with distinct overall survival rates (P < 0.001). Compared with the high-risk group, proportion of naive B cells, plasma cells and CD8+ T cells increased in the low-risk group (P < 0.001). Besides, expressions of HLA and checkpoint genes, such as CD274, CTLA4, LAG3 and TIGIT3, were upregulated in low-risk group. Additionally, highly expressed MGAT5 was validated in breast cancer tissues and cells. Downstream glycosylation products of MGAT5 were all increased in breast cancer. Conclusions: We identified a 19-glycogene signature for risk prediction of breast cancer patients. Patients in the low-risk group demonstrated a higher immune infiltration and better immunotherapy response. The validation of MGAT5 protein suggests a probable pathway and target for the development and treatment of breast cancer.

Desialylated Mesenchymal Stem Cells-Derived Extracellular Vesicles Loaded with Doxorubicin for Targeted Inhibition of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the dominating causes of cancer-related death throughout the world. Treatment options for patients with HCC vary, however, the lack of effective targeted drugs is the major reason for death in advanced HCC patients. In this study, a delivery system based on mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) loaded with doxorubicin (Dox) was developed. In this system, we initially erased terminal linked α2-3 and α2-6 sialic acids on the surface of EVs by neuraminidase. The exhibition of galactose (Gal) and N-acetylgalactosamine (GalNAc) residues in treated MSC-EVs can specifically be recognized by asialoglycoprotein receptor (ASGPR) of hepatoma cells. Compared to free Dox and Dox-loaded EVs, desialylated EVs loaded with Dox significantly presented the improved cellular uptake, prioritized targeting efficacy, and had a better inhibiting effect in vitro and in vivo. Overall, the results of the present study of the demonstrated delivery system using desialylated MSC-EVs suggest its therapeutic potential for HCC.

Dysregulation and prometastatic function of glycosyltransferase C1GALT1 modulated by cHP1BP3/ miR-1-3p axis in bladder cancer.

BACKGROUND: Abnormal glycosylation in a variety of cancer types is involved in tumor progression and chemoresistance. Glycosyltransferase C1GALT1, the key enzyme in conversion of Tn antigen to T antigen, is involved in both physiological and pathological conditions. However, the mechanisms of C1GALT1 in enhancing oncogenic phenotypes and its regulatory effects via non-coding RNA are unclear. METHODS: Abnormal expression of C1GALT1 and its products T antigen in human bladder cancer (BLCA) were evaluated with BLCA tissue, plasma samples and cell lines. Effects of C1GALT1 on migratory ability and proliferation were assessed in YTS-1 cells by transwell, CCK8 and colony formation assay in vitro and by mouse subcutaneous xenograft and trans-splenic metastasis models in vivo. Dysregulated circular RNAs (circRNAs) and microRNAs (miRNAs) were profiled in 3 pairs of bladder cancer tissues by RNA-seq. Effects of miR-1-3p and cHP1BP3 (circRNA derived from HP1BP3) on modulating C1GALT1 expression were investigated by target prediction program, correlation analysis and luciferase reporter assay. Functional roles of miR-1-3p and cHP1BP3 on migratory ability and proliferation in BLCA were also investigated by in vitro and in vivo experiments. Additionally, glycoproteomic analysis was employed to identify the target glycoproteins of C1GALT1. RESULTS: In this study, we demonstrated upregulation of C1GALT1 and its product T antigen in BLCA. C1GALT1 silencing suppressed migratory ability and proliferation of BLCA YTS-1 cells in vitro and in vivo. Subsets of circRNAs and miRNAs were dysregulated in BLCA tissues. miR-1-3p, which is reduced in BLCA tissues, inhibited transcription of C1GALT1 by binding directly to its 3'-untranslated region (3'-UTR). miR-1-3p overexpression resulted in decreased migratory ability and proliferation of YTS-1 cells. cHP1BP3 was upregulated in BLCA tissues, and served as an miR-1-3p "sponge". cHP1BP3 was shown to modulate migratory ability, proliferation, and colony formation of YTS-1 cells, and displayed tumor-suppressing activity in BLCA. Target glycoproteins of C1GALT1, including integrins and MUC16, were identified. CONCLUSIONS: This study reveals the pro-metastatic and proliferative function of upregulated glycosyltransferase C1GLAT1, and provides preliminary data on mechanisms underlying dysregulation of C1GALT1 via miR-1-3p / cHP1BP3 axis in BLCA.

Vesicular Ganglioside GM1 From Breast Tumor Cells Stimulated Epithelial-to-Mesenchymal Transition of Recipient MCF-10A Cells.

Small extracellular vesicles (sEVs) are a type of membrane structure secreted by cells, which are involved in physiological and pathological processes by participating in intercellular communication. Glycosphingolipids (GSLs) are enriched in sEV and can be delivered to recipient cells. In this study, we found that overexpression of B3GALT4, the glycosyltransferase responsible for ganglioside GM1 synthesis, can induce the epithelial-mesenchymal transition (EMT) process in MCF-10A cells. Moreover, GM1 was verified to be presented on sEV from breast cancer cells. Overexpression of B3GALT4 resulted in elevated vesicular GM1 levels and increased sEV secretion in breast cancer cells. Proteomic analysis revealed that eleven sEV secretion-related proteins were differentially expressed, which might contribute to the altered sEV secretion. Of the identified proteins, 15 oncogenic differentially expressed proteins were documented to be presented in sEV. With the treatment of GM1-enriched sEV from breast cancer cells, the EMT process was induced in recipient non-tumorigenic epithelial MCF-10A cells. Our findings demonstrated that GM1-enriched sEVs derived from breast cancer cells induced the EMT process of recipient cells, which might provide essential information on the biological function of vesicular GM1.

Melatonin reduces proliferation and promotes apoptosis of bladder cancer cells by suppressing O-GlcNAcylation of cyclin-dependent-like kinase 5.

Melatonin helps to maintain circadian rhythm, exerts anticancer activity, and plays key roles in regulation of glucose homeostasis and energy metabolism. Glycosylation, a form of metabolic flux from glucose or other monosaccharides, is a common post-translational modification. Dysregulated glycosylation, particularly O-GlcNAcylation, is often a biomarker of cancer cells. In this study, elevated O-GlcNAc level in bladder cancer was inhibited by melatonin treatment. Melatonin treatment inhibited proliferation and migration and enhanced apoptosis of bladder cancer cells. Proteomic analysis revealed reduction in cyclin-dependent-like kinase 5 (CDK5) expression by melatonin. O-GlcNAc modification determined the conformation of critical T-loop domain on CDK5 and further influenced the CDK5 stability. The mechanism whereby melatonin suppressed O-GlcNAc level was based on decreased glucose uptake and metabolic flux from glucose to UDP-GlcNAc, and consequent reduction in CDK5 expression. Melatonin treatment, inhibition of O-GlcNAcylation by OSMI-1, or mutation of key O-GlcNAc site strongly suppressed in vivo tumor growth. Our findings indicate that melatonin reduces proliferation and promotes apoptosis of bladder cancer cells by suppressing O-GlcNAcylation of CDK5.

Integrating transcriptomics, proteomics, glycomics and glycoproteomics to characterize paclitaxel resistance in breast cancer cells.

Chemoresistance is a major factor driving breast cancer (BC) relapse and the high rates of cancer-related deaths. Aberrant levels of glycans are closely correlated with chemoresistance. The essential functions of glycans in chemoresistance is not systematically studied. In this study, an integrated strategy with a combination of transcriptomics, proteomics, glycomics and glycoproteomics was applied to explore the dysregulation of glycogenes, glycan structures and glycoproteins in chemoresistance of breast cancer cells. In paclitaxel (PTX) resistant MCF7 cells, 19 differentially expressed N-glycan-related proteins were identified, of which MGAT4A was the most significantly down-regulated, consistent with decrease in MGAT4A expression at mRNA level in PTX treated BC cells. Glycomic analysis consistently revealed suppressed levels of multi-antennary branching structures using MALDI-TOF/TOF-MS and lectin microarray. Several target glycoproteins bearing suppressed levels of multi-antennary branching structures were identified, and ERK signaling pathway was strongly suppressed in PTX resistant MCF7 cells. Our findings demonstrated the aberrant levels of multi-antennary branching structures and their target glycoproteins on PTX resistance. Systematically integrative multi-omic analysis is expected to facilitate the discovery of the aberrant glycosyltransferases, N-glycosylation and glycoproteins in tumor progression and chemoresistance. SIGNIFICANCE: An integrated strategy with a combination of transcriptomics, proteomics, glycomics and glycoproteomics is crucial to understand the association between glycans and chemoresistance in BC. In this multi-omic analysis, we identified unique glycan-related protein, glycan and glycoprotein signatures defining PTX chemoresistance in BC. This study might provide valuable information to understand molecular mechanisms underlying chemoresistance in BC.

Fucosyltransferase 8 regulation and breast cancer suppression by transcription factor activator protein 2γ.

Alterations of glycosyltransferase expression are often associated with tumor occurrence and progression. Among the many glycosyltransferases, increased expression of fucosyltransferase 8 (FUT8) has been frequently observed to be involved in progression and metastasis of various types of cancer. The regulatory mechanisms of FUT8 expression remain unclear. FUT8 expression was shown, in this study, to be elevated in breast cancer. Systematic analysis revealed that transcription factor activator protein 2γ (AP-2γ) is the target gene of microRNA-10b (miR-10b), which we previously identified as a positive regulator of FUT8. Overexpression of AP-2γ inhibited FUT8 expression, with associated reduction of cell invasiveness and migration ability. AP-2γ was capable of binding to transcription factor STAT3, and phosphorylation of STAT3 induced transcription of the FUT8 gene. On the basis of our findings, we propose that binding of AP-2γ to STAT3 results in formation of the AP-2γ/STAT3 complex and consequent inhibition of STAT3 phosphorylation, thereby preventing entry of p-STAT3 into the nucleus to initiate FUT8 transcription. This study clarifies the molecular mechanisms whereby transcription factor AP-2γ regulates FUT8 expression in breast cancer.

Role of Site-Specific Glycosylation in the I-Like Domain of Integrin ß1 in Small Extracellular Vesicle-Mediated Malignant Behavior and FAK Activation.

Integrin ß1 plays an essential role in the crosstalk between tumor cells and their microenvironment. Aberrant N-glycosylation of integrin ß1 was documented to alter integrin ß1 expression, dimerization, and biological function. However, the biological function of site-specific N-glycosylation of integrin ß1 on extracellular vesicles is not fully understood. In this study, we mutated putative N-glycosylation sites in different domains of integrin ß1. Removal of the N-glycosylation sites on the I-like domain of integrin ß1 (termed the Δ4-6 ß1 mutant) suppressed focal adhesion kinase (FAK) signaling, cell migration, and adhesion compared with other ß1 mutants. Cell adhesion, migration, and activation of FAK were suppressed in recipient MCF7 cells co-cultured with Δ4-6 mutant cells and treated with small extracellular vesicles (sEVs) from Δ4-6 mutant cells. Notably, the wild-type and ß1 mutant were both present in sEVs, and could be transferred to recipient cells via sEVs, resulting in changes of cell behavior. Our findings demonstrate the important roles of N-glycosylation of the I-like domain of integrin ß1. Moreover, the vesicular Δ4-6 ß1 mutant can regulate integrin-mediated functions in recipient cells via sEVs.

High-throughput, in-depth and estimated absolute quantification of plasma proteome using data-independent acquisition/mass spectrometry ("HIAP-DIA").

Mass spectrometry-based plasma proteomics has been demonstrated to be a useful tool capable of quantifying hundreds of proteins in a single LC-MS/MS experiment, for biomarker discovery or elucidation of disease mechanisms. We developed a novel data-independent acquisition (DIA)/MS-based workflow for high-throughput, in-depth and estimated absolute quantification of plasma proteins (termed HIAP-DIA), without depleting high-abundant proteins, in a single-shot experiment. In HIAP-DIA workflow, we generated an ultra-deep cumulative undepleted and depleted spectral library which contained 55,157 peptides and 5,328 proteins, optimized column length (50 cm) and gradient (90 min) of liquid chromatography instrumentation, optimized 50 DIA segments with average isolation window 17 Th, and selected reference proteins for estimated absolute quantification of all plasma proteins. A total of 606 proteins were quantified in triplicate, and 427 proteins were quantified with CV <20% in plasma proteome. R-squared value of overlapped 208 endogenous PQ500 estimated protein amounts from HIAP-DIA and absolute quantification with internal standards was 0.82, indicating high quantification accuracy of HIAP-DIA. As a pilot study, the HIAP-DIA approach described here was applied to a myelodysplastic syndromes (MDS) disease cohort. We achieved absolute quantification of 789 plasma proteins in 22 clinical plasma samples, spanning less than six orders of magnitude with quantification limit 10-20 ng/mL, and discovered 95 differentially expressed proteins providing insights into MDS pathophysiology.

Bisecting GlcNAc modification diminishes the pro-metastatic functions of small extracellular vesicles from breast cancer cells.

Small extracellular vesicles (sEVs) are enriched in glycoconjugates and display specific glycosignatures. Aberrant expression of surface glycoconjugates is closely correlated with cancer progression and metastasis. The essential functions of glycoconjugates in sEVs are poorly understood. In this study, we observed significantly reduced levels of bisecting GlcNAc in breast cancer. Introduction of bisecting GlcNAc into breast cancer cells altered the bisecting GlcNAc status on sEVs, and sEVs with diverse bisecting GlcNAc showed differing functions on recipient cells. Carcinogenesis and metastasis of recipient cells were enhanced by sEVs with low bisecting GlcNAc, and the pro-metastatic functions of sEVs was diminished by high bisecting GlcNAc modification. We further identified vesicular integrin ß1 as a target protein bearing bisecting GlcNAc. Metastasis of recipient cells was strongly suppressed by high bisecting GlcNAc levels on vesicular ß1. Our findings demonstrate the important roles of glycoconjugates on sEVs. Modification of sEV glycosylation may contribute to development of novel targets in breast cancer therapy.

Bisecting N-Acetylglucosamine on EGFR Inhibits Malignant Phenotype of Breast Cancer via Down-Regulation of EGFR/Erk Signaling.

Glycosylation, the most prevalent and diverse post-translational modification of protein, plays crucial biological roles in many physiological and pathological events. Alteration of N-glycan has been detected during breast cancer progression. Among the specific N-glycan structures, bisecting N-Acetylglucosamine (GlcNAc) is a ß1,4-linked GlcNAc attached to the core ß-mannose residue, and is catalyzed by glycosyltransferase MGAT3. Bisecting GlcNAc levels were commonly dysregulated in different types of cancer. In this study, we utilized mass spectrometry and lectin microarray analysis to investigate aberrant N-glycans in breast cancer cells. Our data showed the decreased levels of bisecting GlcNAc and down-regulated expression of MGAT3 in breast cancer cells than normal epithelial cells. Using PHA-E (a plant lectin recognizing and combining bisecting GlcNAc) based enrichment coupled with nanoLC-MS/MS, we analyzed the glycoproteins bearing bisecting GlcNAc in various breast cancer cells. Among the differentially expressed glycoproteins, levels of bisecting GlcNAc on EGFR were significantly decreased in breast cancer cells, confirmed by immunostaining and immunoprecipitation. We overexpressed MGAT3 in breast cancer MDA-MB-231 cells, and overexpression of MGAT3 significantly enhanced the bisecting N-GlcNAc on EGFR and suppressed the EGFR/Erk signaling, which further resulted in the reduction of migratory ability, cell proliferation, and clonal formation. Taken together, we conclude that bisecting N-GlcNAc on EGFR inhibits malignant phenotype of breast cancer via down-regulation of EGFR/Erk signaling.