Comparison of different fractionation strategies for in-depth phosphoproteomics by liquid chromatography tandem mass spectrometry.

Phosphorylation, a major posttranslational modification of proteins, plays an important role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because of its low abundance and the fact that the analysis can be hindered by the presence of highly abundant non-phosphoproteins. In order to reduce the sample complexity and improve the efficiency of identification of phosphopeptides, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) was utilized to enrich phosphopeptides from a murine macrophage cell lysate. Strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC), and solution isoelectric focusing (sIEF) were investigated in detail for phosphopeptide fractionation strategies followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from the cell lysates in three fractionation approaches. The SCX fractionation contained the largest number of phosphoproteins and phosphopeptides that were identified. In addition, 4336, 2064, and 2424 phosphopeptides were identified from SCX-LC-MS/MS, ERLIC-LC-MS/MS, and sIEF-LC/MS-MS, including 2430, 438, and 751 phosphopeptides that were only specifically found in SCX, ERLIC, and sIEF fractionations. In conclusion, these three fractionation strategies demonstrated great complementarity, which greatly improved the efficiency of identification of phosphopeptides and can be suitable for use in in-depth phosphoproteome research. Graphical Abstract.

Sialylation of vasorin by ST3Gal1 facilitates TGF-ß1-mediated tumor angiogenesis and progression.

ST3Gal1 is a key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF-ß1, as a potential candidate that links ST3Gal1 to angiogenesis. LC-MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O-glycans are sialyl-3T and disialyl-T. ST3GAL1-silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF-ß1 by 2- to 3-fold and thereby dampening TGF-ß1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse-free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14-7.67). Since TGF-ß1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-ß1 upregulates ST3Gal1 to circumvent the negative impact of VASN.

Autophagic degradation of SQSTM1 inhibits ovarian cancer motility by decreasing DICER1 and AGO2 to induce MIRLET7A-3P.

The relationship between macroautophagy/autophagy and miRNA in regulating cancer cell motility is not clearly delineated. Here, we found that induction of BECN1-dependent or -independent autophagy decreased ubiquitin-binding proteins SQSTM1/p62 and CALCOCO2/NDP52. Downregulation of SQSTM1 (but not CALCOCO2) led to a decrease of the miRNA-processing enzyme DICER1 and the miRNA effector AGO2. The autophagy-mediated reduction of levels of SQSTM1, DICER1 or AGO2 resulted in increased MIRLET7A-3P (but not MIRLET7A-5P or PRE-MIRLET7A miRNA) and suppressed ovarian cancer motility. The investigation of the MIRLET7A effects on cancer cell motility showed that synthetic MIRLET7A-3P (3 nM) inhibited, whereas MIRLET7A-5P (100 nM) increased cancer cell motility. Moreover, downregulation of MIRLET7A-3P with antisense of MIRLET7A-3P miRNA (MIRLET7A-3P inhibitor; 3 nM) reversed the nutrient depletion- and rVP1-mediated suppression of ovarian cancer cell motility. In addition, restoring SQSTM1, DICER1 and AGO2 with inhibition of autophagic degradation or overexpression of DICER1 and AGO2 reversed the autophagy-associated enhancement of MIRLET7A-3P and inhibition of motility. Examination of ovarian cancer tissue microarray further showed that the levels of SQSTM1, DICER1 and AGO2 in the tumor were higher than those in the non-tumor cells and negatively correlated with the levels of autophagy and MIRLET7A-3P. Our results demonstrated that induction of autophagy to decrease SQSTM1, DICER1 and AGO2 and increase MIRLET7A-3P is a potential therapeutic strategy for suppressing ovarian cancer cell motility. Abbreviations: ACTB: actin beta; AGO2: argonaute 2, RISC catalytic component; ATG: autophagy related; BCIP/NBT: 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium; BECN1: beclin 1, autophagy related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; DICER1: dicer 1, ribonuclease III; EBSS: Earle balanced salt solution; FBS: fetal bovine serum; HGF: hepatocyte growth factor; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MIRLET7A: microRNA LET-7A: MIR16: microRNA 16; MIR29C: microRNA 29C; miRNA: microRNA; MMP: matrix metallopeptidase; PRE-MIRNA: precursor microRNA; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RISC: RNA-induced silencing complex; rVP1: recombinant foot-and-mouth disease virus capsid protein VP1; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; WIPI: WD repeat domain, phosphoinositide interacting.

Reciprocal feedback regulation of ST3GAL1 and GFRA1 signaling in breast cancer cells.

GFRA1 and RET are overexpressed in estrogen receptor (ER)-positive breast cancers. Binding of GDNF to GFRA1 triggers RET signaling leading to ER phosphorylation and estrogen-independent transcriptional activation of ER-dependent genes. Both GFRA1 and RET are membrane proteins which are N-glycosylated but no O-linked sialylation site on GFRA1 or RET has been reported. We found GFRA1 to be a substrate of ST3GAL1-mediated O-linked sialylation, which is crucial to GDNF-induced signaling in ER-positive breast cancer cells. Silencing ST3GAL1 in breast cancer cells reduced GDNF-induced phosphorylation of RET, AKT and ERα, as well as GDNF-mediated cell proliferation. Moreover, GDNF induced transcription of ST3GAL1, revealing a positive feedback loop regulating ST3GAL1 and GDNF/GFRA1/RET signaling in breast cancers. Finally, we demonstrated ST3GAL1 knockdown augments anti-cancer efficacy of inhibitors of RET and/or ER. Moreover, high expression of ST3GAL1 was associated with poor clinical outcome in patients with late stage breast cancer and high expression of both ST3GAL1 and GFRA1 adversely impacted outcome in those with high grade tumors.

High expression FUT1 and B3GALT5 is an independent predictor of postoperative recurrence and survival in hepatocellular carcinoma.

Cancer may arise from dedifferentiation of mature cells or maturation-arrested stem cells. Previously we reported that definitive endoderm from which liver was derived, expressed Globo H, SSEA-3 and SSEA-4. In this study, we examined the expression of their biosynthetic enzymes, FUT1, FUT2, B3GALT5 and ST3GAL2, in 135 hepatocellular carcinoma (HCC) tissues by qRT-PCR. High expression of either FUT1 or B3GALT5 was significantly associated with advanced stages and poor outcome. Kaplan Meier survival analysis showed significantly shorter relapse-free survival (RFS) for those with high expression of either FUT1 or B3GALT5 (P = 0.024 and 0.001, respectively) and shorter overall survival (OS) for those with high expression of B3GALT5 (P = 0.017). Combination of FUT1 and B3GALT5 revealed that high expression of both genes had poorer RFS and OS than the others (P < 0.001). Moreover, multivariable Cox regression analysis identified the combination of B3GALT5 and FUT1 as an independent predictor for RFS (HR: 2.370, 95% CI: 1.505-3.731, P < 0.001) and OS (HR: 2.153, 95% CI: 1.188-3.902, P = 0.012) in HCC. In addition, the presence of Globo H, SSEA-3 and SSEA-4 in some HCC tissues and their absence in normal liver was established by immunohistochemistry staining and mass spectrometric analysis.

From the Cover: Comparative Proteomics Reveals Silver Nanoparticles Alter Fatty Acid Metabolism and Amyloid Beta Clearance for Neuronal Apoptosis in a Triple Cell Coculture Model of the Blood-Brain Barrier.

Silver nanoparticles (AgNPs) enter the central nervous system through the blood-brain barrier (BBB). AgNP exposure can increase amyloid beta (Aß) deposition in neuronal cells to potentially induce Alzheimer's disease (AD) progression. However, the mechanism through which AgNPs alter BBB permeability in endothelial cells and subsequently lead to AD progression remains unclear. This study investigated whether AgNPs disrupt the tight junction proteins of brain endothelial cells, and alter the proteomic metabolism of neuronal cells underlying AD progression in a triple cell coculture model constructed using mouse brain endothelial (bEnd.3) cells, mouse brain astrocytes (ALT), and mouse neuroblastoma neuro-2a (N2a) cells. The results showed that AgNPs accumulated in ALT and N2a cells because of the disruption of tight junction proteins, claudin-5 and ZO-1, in bEnd.3 cells. The proteomic profiling of N2a cells after AgNP exposure identified 298 differentially expressed proteins related to fatty acid metabolism. Particularly, AgNP-induced palmitic acid production was observed in N2a cells, which might promote Aß generation. Moreover, AgNP exposure increased the protein expression of amyloid precursor protein (APP) and Aß generation-related secretases, PSEN1, PSEN2, and ß-site APP cleaving enzyme for APP cleavage in ALT and N2a cells, stimulated Aß40 and Aß42 secretion in the culture medium, and attenuated the gene expression of Aß clearance-related receptors, P-gp and LRP-1, in bEnd.3 cells. Increased Aß might further aggregate on the neuronal cell surface to enhance the secretion of inflammatory cytokines, MCP-1 and IL-6, thus inducing apoptosis in N2a cells. This study suggested that AgNP exposure might cause Aß deposition and inflammation for subsequent neuronal cell apoptosis to potentially induce AD progression.

Interaction of glycosphingolipids GD3 and GD2 with growth factor receptors maintains breast cancer stem cell phenotype.

Many studies have suggested that disialogangliosides, GD2 and GD3, are involved in the development of various tumor types. However, the functional relationships between ganglioside expression and cancer development or aggressiveness are not fully described. GD3 is upregulated in approximately half of all invasive ductal breast carcinoma cases, and enhanced expression of GD3 synthase (GD3S, alpha-N-acetylneuraminide alpha-2,8-sialyltransferase) in estrogen receptor-negative breast tumors, was shown to correlate with reduced overall patient survival. We previously found that GD2 and GD3, together with their common upstream glycosyltransferases, GD3S and GD2/GM2 synthase, maintain a stem cell phenotype in breast cancer stem cells (CSCs). In the current study, we demonstrate that GD3S alone can sustain CSC properties and also promote malignant cancer properties. Using MALDI-MS and flow cytometry, we found that breast cancer cell lines, of various subtypes with or without ectopic GD3S-expression, exhibited distinct GD2/GD3 expression profiles. Furthermore, we found that GD3 was associated with EGFR and activated EGFR signaling in both breast CSCs and breast cancer cell lines. In addition, GD3S knockdown enhanced cytotoxicity of the EGFR-inhibitor gefitinib in resistant MDA-MB468 cells, both in vitro and in vivo. Based on this evidence, we propose that GD3S contributes to gefitinib-resistance in EGFR-positive breast cancer cells and may be an effective therapeutic target in drug-resistant breast cancers.

Glycosphingolipid dynamics in human embryonic stem cell and cancer: their characterization and biomedical implications.

Glycosphingolipids (GSLs) are composed of complex glycans linked to sphingosines and various fatty acid chains. Antibodies against several GSLs designated as stage-specific embryonic antigens (SSEAs), have been widely used to characterize differentiation of embryonic stem (ES) cells. In view of the cross-reactivities of these antibodies with multiple glycans, a few laboratories have employed advanced mass spectrometry (MS) technologies to define the dynamic changes of surface GSLs upon ES differentiation. However, the amphiphilic nature and heterogeneity of GSLs make them difficult to decipher. In our studies, systematic survey of GSL expression profiles in human ES cells and differentiated derivatives was conducted, primarily with matrix-assisted laser desorption/ionization MS (MALDI-MS) and MS/MS analyses. In addition to the well-known ES-specific markers, SSEA-3 and SSEA-4, several previously undisclosed globo- and lacto-series GSLs, including Gb4Cer, Lc4Cer, fucosyl Lc4Cer, Globo H, and disialyl Gb5Cer were identified in the undifferentiated human ES and induced pluripotent stem cells. Furthermore, during differentiation to embryoid body outgrowth, the core structures of GSLs switched from globo- and lacto- to ganglio-series. Lineage-specific differentiation was also marked by alterations of specific GSLs. During differentiation into neural progenitors, core structures shifted to primarily ganglio-series dominated by GD3. GSL patterns shifted to prominent expression of Gb4Cer with little SSEA-3 and- 4 or GD3 during endodermal differentiation. Several issues relevant to MS analysis and novel GSLs in ES cells were discussed. Finally, unique GSL signatures in ES and cancer cells are exploited in glycan-targeted anti-cancer immunotherapy and their mechanistic investigations were discussed using anti-GD2 mAb and Globo H as examples.

Alterations of Glycosphingolipids in Embryonic Stem Cell Differentiation and Development of Glycan-Targeting Cancer Immunotherapy.

All mammalian cells display an array of surface glycans that can modulate cellular interactions and regulate the development of an organism. In spite of their important implications in health and disease, investigations of glycans pose a great challenge given their inherent heterogeneity and the frequently observed cross-reactivities of antibodies against glycosphingolipids (GSLs) with multiple glycans, which may lead to erroneous interpretation and conclusion. We employed matrix-assisted laser desorption-ionization mass spectrometry and tandem MS/MS analyses to systematically delineate changes in GSLs during differentiation of human embryonic stem (ES) and induced pluripotent stem cells into various derivatives. In addition to the well-known human ES-specific markers, stage-specific embryonic antigen (SSEA)-3 and SSEA-4 and several globo- and lacto-series GSLs (Gb4Cer, Lc4Cer, fucosyl-Lc4Cer, Globo H, and disialyl-Gb5Cer) not reported previously were identified. A close association of the switching of core structures of GSLs from globo- and lacto- to ganglio-series during ES differentiation was revealed, consistent with the observed changes in GSL expression during murine embryonic development. Such switching was accompanied with the concerted changes in the expression of glycosyltransferases during differentiation. Finally, since aberrant glycosylation is a general feature of cancer, targeting tumor-associated surface glycans for cancer immunotherapy is gaining international attention. While GD2-targeted immunotherapy of neuroblastoma represents the first antiglycan monoclonal antibody to obtain FDA approval for standard medical care, Globo H-targeted immunotherapy of breast cancer has generated promising results in the ongoing multinational clinical trials. In this study, we also describe the studies of the scientific rationales for the use of glycans as anticancer immunotherapeutics.

Fucosylation of LAMP-1 and LAMP-2 by FUT1 correlates with lysosomal positioning and autophagic flux of breast cancer cells.

Alpha1,2-fucosyltransferases, FUT1 and FUT2, which transfer fucoses onto the terminal galactose of N-acetyl-lactosamine via α1,2-linkage have been shown to be highly expressed in various types of cancers. A few studies have shown the involvement of FUT1 substrates in tumor cell proliferation and migration. Lysosome-associated membrane protein 1, LAMP-1, has been reported to carry alpha1,2-fucosylated Lewis Y (LeY) antigens in breast cancer cells, however, the biological functions of LeY on LAMP-1 remain largely unknown. Whether or not its family member, LAMP-2, displays similar modifications and functions as LAMP-1 has not yet been addressed. In this study, we have presented evidence supporting that both LAMP-1 and 2 are substrates for FUT1, but not FUT2. We have also demonstrated the presence of H2 and LeY antigens on LAMP-1 by a targeted nanoLC-MS(3) and the decreased levels of fucosylation on LAMP-2 by MALDI-TOF analysis upon FUT1 knockdown. In addition, we found that the expression of LeY was substantial in less invasive ER+/PR+/HER- breast cancer cells (MCF-7 and T47D) but negligible in highly invasive triple-negative MDA-MB-231 cells, of which LeY levels were correlated with the levels of LeY carried by LAMP-1 and 2. Intriguingly, we also observed a striking change in the subcellular localization of lysosomes upon FUT1 knockdown from peripheral distribution of LAMP-1 and 2 to a preferential perinuclear accumulation. Besides that, knockdown of FUT1 led to an increased rate of autophagic flux along with diminished activity of mammalian target of rapamycin complex 1 (mTORC1) and enhanced autophagosome-lysosome fusion. This may be associated with the predominantly perinuclear distribution of lysosomes mediated by FUT1 knockdown as lysosomal positioning has been reported to regulate mTOR activity and autophagy. Taken together, our results suggest that downregulation of FUT1, which leads to the perinuclear localization of LAMP-1 and 2, is correlated with increased rate of autophagic flux by decreasing mTOR signaling and increasing autolysosome formation.

PATZ1 induces PP4R2 to form a negative feedback loop on IKK/NF-κB signaling in lung cancer.

Activation of IKK enhances NF-κB signaling to facilitate cancer cell migration, invasion and metastasis. Here, we uncover the existence of a negative feedback loop of IKK. The transcription factor PATZ1 induces protein phosphatase-4 (PP4) regulatory subunit 2 (PP4R2) in an IKK-dependent manner. PP4R2 enhances the binding of PP4 to phosphorylated IKK to inactivate IKK/NF-κB signaling during sustained stimulation by cellular stimuli such as growth factors and inflammatory mediators. Matched pair studies reveal that primary lung cancers express more PATZ1 and PP4R2 than lymph node metastases in patients. Ectopic PATZ1 decreases invasion/colonization of lung cancers and prolongs the survival of xenograft mice. These effects of PATZ1 are reversed by downregulating PP4R2. Our results suggest that PATZ1 and PP4R2 provide negative feedback on IKK/NF-κB signaling to prevent cancer cells from over-stimulation from cellular stimuli; a decline in PATZ1 and PP4R2 is functionally associated with cancer migration/invasion and agents enhancing PATZ1 and PP4R2 are worth exploring to prevent invasion/metastasis of lung cancers.

Analysis of dermal papilla cell interactome using STRING database to profile the ex vivo hair growth inhibition effect of a vinca alkaloid drug, colchicine.

Dermal papillae (DPs) control the formation of hair shafts. In clinical settings, colchicine (CLC) induces patients' hair shedding. Compared to the control, the ex vivo hair fiber elongation of organ cultured vibrissa hair follicles (HFs) declined significantly after seven days of CLC treatment. The cultured DP cells (DPCs) were used as the experimental model to study the influence of CLC on the protein dynamics of DPs. CLC could alter the morphology and down-regulate the expression of alkaline phosphatase (ALP), the marker of DPC activity, and induce IκBα phosphorylation of DPCs. The proteomic results showed that CLC modulated the expression patterns (fold>2) of 24 identified proteins, seven down-regulated and 17 up-regulated. Most of these proteins were presumably associated with protein turnover, metabolism, structure and signal transduction. Protein-protein interactions (PPI) among these proteins, established by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, revealed that they participate in protein metabolic process, translation, and energy production. Furthermore, ubiquitin C (UbC) was predicted to be the controlling hub, suggesting the involvement of ubiquitin-proteasome system in modulating the pathogenic effect of CLC on DPC.

MIG-7 and phosphorylated prohibitin coordinately regulate lung cancer invasion/metastasis.

Growth factors and COX-2/PGE2 enhance lung cancer invasion/metastasis via PI3K/Akt and RAS/Raf. Here, we explored their mechanism of action further. We found first that higher levels of migration inducting gene-7 protein (MIG-7) and PHB phosphorylated at threonine 258 (phospho-PHBT258) are positively correlated with advanced stages of human lung cancer in tissue microarray. PGE2 or growth factors such as EGF, HGF and IGF-1 increased complex formation of phospho-PHBT258 with Ras, phospho-AktS473, phospho-Raf-1S338, MEKK1 and IKKα/ßS176/180 in the raft domain transiently within 1 hour and MIG-7 in the cytosol 12-24 hours later. Association of phospho-PHBT258 with MEKK1 but not MEKK3 activates IKK/IκB/NF-κB and MEK/ERK to increase cellular COX-2/PGE2 and an E-cadherin suppressor Snail leading to enhancement of epithelial-mesenchymal transition (EMT) and lung cancer migration/invasion. MIG-7, on the other hand, was induced by growth factors and PGE2 via Akt/GSK-3ß in a phospho-PHBT258 independent manner. MIG-7 increased two E-cadherin suppressors ZEB-1 and Twist to enhance EMT and cancer migration/invasion. Downregulating phospho-PHBT258 and MIG-7 had an additive effect on attenuating lung cancer invasion/metastasis and prolonging the survival of xenograft mice. Phospho-PHBT258 and MIG-7 may thus play complementary roles in the initiation and sustainment of the effects of growth factors and COX-2/PGE2 on cancer invasion/metastasis.

Recombinant viral capsid protein VP1 suppresses lung cancer metastasis by inhibiting COX-2/PGE2 and MIG-7.

Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus binds to integrins to modulate Akt/GSK3-ß signaling and suppress migration/invasion and metastasis of cancer cells, but the underlying molecular mechanism is unclear. Here, we showed that the rVP1-mediated inhibition of Akt/GSK3-ß signaling and cell migration/invasion was accompanied by downregulation in phosphatidylinositol (3,4,5)-triphosphate (PIP3), integrin-linked kinase (ILK) and IKK/NF-κB signaling as well as suppression of COX-2/PGE2 and MIG-7. Addition of PIP3 or overexpression of ILK reversed the rVP1-induced inhibition of IKK/NF-κB signaling, COX-2 and MIG-7. The rVP1-mediated downregulation of COX-2/PGE2 and MIG-7 led to not only attenuation of epithelial-mesenchymal transition, MMP2 activity and invasion of lung cancer cells in vitro but also decreased tumor growth and metastasis of lung cancer in xenograft mice. Moreover, downregulation of COX-2/PGE2 and MIG-7 significantly prolonged the overall and disease-free survival of lung cancer-bearing mice. These results suggest that rVP1 inhibits cancer invasion/metastasis, partly if not mainly, via downregulating integrin/PI3K/Akt, ILK and IKK/NF-κB signaling to suppress expression of COX-2/PGE2 and MIG-7.

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Increasing the depth of mass spectrometry-based glycomic coverage by additional dimensions of sulfoglycomics and target analysis of permethylated glycans.

Hog or porcine gastric mucin resembles the human source in carrying not only blood group antigens but also the rather rare α4-GlcNAc-capped terminal epitope functionally implicated in protection against Helicobacter pylori infection. Being more readily available and reasonably well characterized, it serves as a good reagent for immunobiological studies, as well as a standard for analytical methodology developments. Current approaches in mass spectrometry (MS)-based glycomic mapping remain vastly inadequate in revealing the full complexity of glycosylation, particularly for cases such as the extremely heterogeneous O-glycosylation of mucosal mucins that can be further sulfated. We demonstrate here a novel concerted workflow that extends the conventional matrix-assisted laser desorption/ionization­mass spectrometry (MALDI-MS) mapping of permethylated glycans in positive ion mode to include a further step of sulfoglycomic analysis in negative ion mode. This was facilitated by introducing a mixed-mode solid-phase extraction step, which allows direct cleanup and simultaneous fractionation of the permethylated glycans into separate non-sulfated and sulfated pools in one single step. By distinct MALDI-MS/MS fragmentation patterns, all previously known structural features of porcine gastric mucin including the terminal epitopes and location of sulfates could be readily defined. We additionally showed that both arms of the core 2 structures could be extended via 6-O-sulfated GlcNAc to yield a series of disulfated O-glycans not previously reported, thus expanding its current glycomic coverage. However, a targeted LC-MSn analysis was required and best suited to dig even deeper into validating the occurrence of very minor structural isomers carrying the Lewis Y epitope implicated by positive antibody binding.

Pre-existing Fas ligand (FasL) in cancer cells elicits tumor-specific protective immunity, but delayed induction of FasL expression after inoculation facilitates tumor formation.

Overexpression of Fas ligand (FasL) in cancer cells elicits potential antitumor effects via recruitment of neutrophils. Conversely, FasL-expressing tumors may counterattack tumor-infiltrating lymphocytes by delivering apoptotic death signals via Fas/FasL interactions, which may lead to tumor escape. In order to distinguish the role of FasL in antitumor activity and tumor progression, Lewis lung carcinoma cells (LLC-1) were used to establish the cell line LLC-FasL, in which FasL expression was repressed by doxycycline (Dox) treatment and induced in the absence of Dox. LLC-FasL cells promote tumor regression when expressing FasL, whereas tumor outgrowth is observed by depletion of FasL expression. To investigate whether initial expression of FasL during tumor formation is critical for FasL-mediated tumor regression, Dox-treated LLC-FasL cells were inoculated into Dox-treated mice, but Dox treatment was stopped 5 days after inoculation. When low cell numbers were inoculated, we observed 80% survival and no tumor formation, whereas no mice survived inoculation with high cell numbers, despite the delayed induction of FasL by Dox withdrawal. The inoculation of a high density of cells may establish a favorable tumor microenvironment before the expression of FasL. Our findings demonstrate that FasL may elicit antitumor activity when it is initially present on injected cancer cells and thus can act prior to tumor microenvironment formation. Furthermore, a well-established tumor microenvironment abrogates FasL-mediated antitumor activity.

MIG-7 controls COX-2/PGE2-mediated lung cancer metastasis.

More effective treatments for metastatic lung cancer remain a pressing clinical need. In this study, we identified migration inducting gene-7 (MIG-7) protein as critical for COX-2/prostaglandin E2 (PGE2)- and Akt/GSK-3ß-dependent tumor invasion/metastasis. COX-2/PGE2 activated EP4 to enhance Akt and GSK-3ß phosphorylation and ß-catenin/T-cell factor/lymphoid enhancer factor signaling leading to MIG-7 upregulation. RNAi-mediated attenuation of MIG-7 blocked COX-2/PGE2- and Akt/GSK-3ß-mediated migration/invasion effects. Furthermore, MIG-7 protein inhibited protein phosphatase 2A to sustain Akt/GSK-3ß phosphorylation and cancer-cell migration/invasion. Cancer cells overexpressing MIG-7 exhibited increased expression of ZEB-1 and Twist in parallel with epithelial-mesenchymal transition, metastasis and cancer lethality. MIG-7 protein level positively correlated with advanced stages of human lung cancers. MIG-7 thus offers a theranostic target for cancer metastases arising from aberrant activation of the cellular COX-2/PGE2 and Akt/GSK-3ß signaling pathways.