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.

The K346T mutant of GnT-III bearing weak in vitro and potent intracellular activity.

BACKGROUND: N-Acetylglucosaminyltransferase-III (GnT-III, also designated MGAT3) catalyzes the formation of a specific N-glycan branch, bisecting GlcNAc, in the Golgi apparatus. Bisecting GlcNAc is a key residue that suppresses N-glycan maturation and is associated with the pathogenesis of cancer and Alzheimer's disease. However, it remains unclear how GnT-III recognizes its substrates and how GnT-III activity is regulated in cells. METHODS: Using AlphaFold2 and structural comparisons, we predicted the key amino acid residues in GnT-III that interact with substrates in the catalytic pocket. We also performed in vitro activity assay, lectin blotting analysis and N-glycomic analysis using point mutants to assess their activity. RESULTS: Our data suggested that E320 of human GnT-III is the catalytic center. More interestingly, we found a unique mutant, K346T, that exhibited lower in vitro activity and higher intracellular activity than wild-type GnT-III. The enzyme assays using various substrates showed that the substrate specificity of K346T was unchanged, whereas cycloheximide chase experiments revealed that the K346T mutant has a slightly shorter half-life, suggesting that the mutant is unstable possibly due to a partial misfolding. Furthermore, TurboID-based proximity labeling showed that the localization of the K346T mutant is shifted slightly to the cis side of the Golgi, probably allowing for prior action to competing galactosyltransferases. CONCLUSIONS: The slight difference in K346T localization may be responsible for the higher biosynthetic activity despite the reduced activity. GENERAL SIGNIFICANCE: Our findings underscore the importance of fine intra-Golgi localization and reaction orders of glycosyltransferases for the biosynthesis of complex glycan structures in cells.

Glycoproteomic analysis of regulatory effects of bisecting N-glycans on N-glycan biosynthesis and protein expressions in human HK-2 cells.

Bisecting N-glycan is known to be a metastasis suppressor and plays a regulatory role in the biosynthesis of N-glycans. Previous studies have shown that bisecting N-glycans are capable of modulating both the branching and terminal modifications of glycans. However, these effects have been investigated mainly by glycomic approaches and it remains unclear how they alter when glycans are attached to different glycosites of proteins. Here, we systematically investigated the regulatory roles of bisecting N-glycans in human HK-2 cells using StrucGP, a strategy we developed for structural interpretation of site-specific N-glycans on glycoproteins. The glycoproteomics analysis showed that most of bisecting N-glycans are complex type and often occur in company with core fucosylation. With the overexpression and knockdown of MGAT3, the only enzyme responsible for bisecting N-glycan synthesis, we found that bisecting N-glycans can impact the biosynthesis of N-glycans from multiple aspects, including glycan types, branching, sialylation, fucosylation (different effects for core and terminal fucosylation) as well as the presence of terminal N-acetylglucosamine. Furthermore, gene ontology analysis suggested that most proteins with bisecting N-glycans located in the extracellular region or membrane, where they function mostly in cell adhesion, extracellular matrix regulation and cell signaling. Finally, we showed that overexpression of bisecting N-glycans had a broad impact on the protein expressions of HK-2 cells, involving multiple biological processes. Taken together, our work systematically demonstrated the expression profiles of bisecting N-glycans, and their regulatory effects on the biosynthesis of N-glycans and protein expressions, which provide valuable information for the functional elucidation of bisecting N-glycans.

Integrated glycomics and genetics analyses reveal a potential role for N-glycosylation of plasma proteins and IgGs, as well as the complement system, in the development of type 1 diabetes.

AIMS/HYPOTHESIS: We previously demonstrated that N-glycosylation of plasma proteins and IgGs is different in children with recent-onset type 1 diabetes compared with their healthy siblings. To search for genetic variants contributing to these changes, we undertook a genetic association study of the plasma protein and IgG N-glycome in type 1 diabetes. METHODS: A total of 1105 recent-onset type 1 diabetes patients from the Danish Registry of Childhood and Adolescent Diabetes were genotyped at 183,546 genetic markers, testing these for genetic association with variable levels of 24 IgG and 39 plasma protein N-glycan traits. In the follow-up study, significant associations were validated in 455 samples. RESULTS: This study confirmed previously known plasma protein and/or IgG N-glycosylation loci (candidate genes MGAT3, MGAT5 and ST6GAL1, encoding beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase, alpha-1,6-mannosylglycoprotein 6-beta-N-acetylglucosaminyltransferase and ST6 beta-galactoside alpha-2,6-sialyltransferase 1 gene, respectively) and identified novel associations that were not previously reported for the general European population. First, novel genetic associations of IgG-bound glycans were found with SNPs on chromosome 22 residing in two genomic intervals close to candidate gene MGAT3; these include core fucosylated digalactosylated disialylated IgG N-glycan with bisecting N-acetylglucosamine (GlcNAc) (pdiscovery=7.65 × 10-12, preplication=8.33 × 10-6 for the top associated SNP rs5757680) and core fucosylated digalactosylated glycan with bisecting GlcNAc (pdiscovery=2.88 × 10-10, preplication=3.03 × 10-3 for the top associated SNP rs137702). The most significant genetic associations of IgG-bound glycans were those with MGAT3. Second, two SNPs in high linkage disequilibrium (missense rs1047286 and synonymous rs2230203) located on chromosome 19 within the protein coding region of the complement C3 gene (C3) showed association with the oligomannose plasma protein N-glycan (pdiscovery=2.43 × 10-11, preplication=8.66 × 10-4 for the top associated SNP rs1047286). CONCLUSIONS/INTERPRETATION: This study identified novel genetic associations driving the distinct N-glycosylation of plasma proteins and IgGs identified previously at type 1 diabetes onset. Our results highlight the importance of further exploring the potential role of N-glycosylation and its influence on complement activation and type 1 diabetes susceptibility.

Glycoproteomic analysis reveals the effects of bisecting GlcNAc in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is the second major subtype of primary liver cancer and has caused more and more attention with increasing incidence and mortality worldwide. Our previous study found that bisecting N-glycans are commonly increased in ICC, while the effects and potential functions of bisecting GlcNAc in ICC are still largely unclear. In this study, we further confirmed that the structures of bisecting GlcNAc were significantly up-regulated in ICC compared with paracancer tissues by glycoproteomic data and lectin histochemistry. The expression of its glycosyltransferase MGAT3 was also up-regulated in ICC tissues at both mRNA and protein levels, and expression of MGAT3 is negatively correlated with overall survival explored by bioinformatic analyses and published datasets from 255 patients. Next, the silencing of MGAT3 could inhibit the growth and invasion of ICC cells, and overexpressing of MGAT3 only promoted ICC cell invasion. Further glycoproteomic analysis showed that the commonly glycoproteins modified by bisecting GlcNAc after MGAT3-overexpression in two ICC cell lines were mainly involved in cell movement-related biological processes, such as cell adhesion, integrin-related and ECM-receptor interaction. This study sheds light on the potential effects of bisecting GlcNAc in ICC cells and suggests that MGAT3 might be used as a potential target in the therapy of ICC.

Development of tricyclic N-benzyl-4-hydroxybutanamide derivatives as inhibitors of GABA transporters mGAT1-4 with anticonvulsant, antinociceptive, and antidepressant activity.

γ-Aminobutyric acid (GABA) neurotransmission has a significant impact on the proper functioning of the central nervous system. Numerous studies have indicated that inhibitors of the GABA transporters mGAT1-4 offer a promising strategy for the treatment of several neurological disorders, including epilepsy, neuropathic pain, and depression. Following our previous results, herein, we report the synthesis, biological evaluation, and structure-activity relationship studies supported by molecular docking and molecular dynamics of a new series of N-benzyl-4-hydroxybutanamide derivatives regarding their inhibitory potency toward mGAT1-4. This study allowed us to identify compound 23a (N-benzyl-4-hydroxybutanamide bearing a dibenzocycloheptatriene moiety), a nonselective GAT inhibitor with a slight preference toward mGAT4 (pIC50 = 5.02 ± 0.11), and compound 24e (4-hydroxy-N-[(4-methylphenyl)-methyl]butanamide bearing a dibenzocycloheptadiene moiety) with relatively high inhibitory activity toward mGAT2 (pIC50 = 5.34 ± 0.09). In a set of in vivo experiments, compound 24e successively showed predominant anticonvulsant activity and antinociception in the formalin model of tonic pain. In contrast, compound 23a showed significant antidepressant-like properties in mice. These results were consistent with the available literature data, which indicates that, apart from seizure control, GABAergic neurotransmission is also involved in the pathophysiology of several psychiatric diseases, however alternative mechanisms underlying this action cannot be excluded. Finally, it is worth noting that the selected compounds showed unimpaired locomotor skills that have been indicated to give reliable results in behavioral assays.

Neuroprotective Effects of Fluoxetine on Molecular Markers of Circadian Rhythm, Cognitive Deficits, Oxidative Damage, and Biomarkers of Alzheimer's Disease-Like Pathology Induced under Chronic Constant Light Regime in Wistar Rats.

There is mounting evidence of circadian rhythm disruption in Alzheimer's disease (AD); however, the cause-and-effect relationship between them is not understood. Chronic constant light exposure effectively disrupts circadian rhythm in rats. On the basis of previous publications, we hypothesized that chronic constant light exposure might contribute significantly to development of AD-like-phenotype in rats and that fluoxetine (Flx) treatment might protect the brain against it. Adult male rats were exposed to normal light-dark cycles, constant light (LL), constant dark, and LL+Flx (5 mg/kg/day, ZT5) for four months. The expression of molecular markers of circadian rhythm: Per2 transcripts; and protein expression of peroxiredoxin-1 (PRX1) and hyperoxidized peroxiredoxins (PRX-SO2/3) were significantly dysregulated in the suprachiasmatic nuclei (SCN) of LL rats, which was prevented with concomitant fluoxetine administration. The levels of glutamate and γ-aminobutyric acid were dysregulated, and oxidative damage was observed in the SCN and hippocampi of LL rats. Fluoxetine treatment conferred protection against oxidative damage in LL rats. Constant light exposure also impaired rats' performance on Y-maze, Morris maze, and novel object recognition test, which was prevented with fluoxetine administration. A significant elevation in soluble Aß1-42 levels, which strongly correlated with upregulation of Bace1 and Mgat3 transcripts was observed in the hippocampus of LL rats. Further, the expression of antiaging gene Sirt1 was downregulated, and neuronal damage indicator Prokr2 was upregulated in hippocampus. Fluoxetine rescued Aß1-42 upregulation and AD-related genes' dysregulation. Our findings show that circadian disruption by exposure to chronic constant light may contribute to progression of AD, which can be prevented with fluoxetine treatment.

Diseases related to Notch glycosylation.

The Notch receptors are a family of transmembrane proteins that mediate direct cell-cell interactions and control numerous cell-fate specifications in humans. The extracellular domains of mammalian Notch proteins contain 29-36 tandem epidermal growth factor-like (EGF) repeats, most of which have O-linked glycan modifications: O-glucose added by POGLUT1, O-fucose added by POFUT1 and elongated by Fringe enzymes, and O-GlcNAc added by EOGT. The extracellular domain is also N-glycosylated. Mutations in the glycosyltransferases modifying Notch have been identified in several diseases, including Dowling-Degos Disease (haploinsufficiency of POFUT1 or POGLUT1), a form of limb-girdle muscular dystrophy (autosomal recessive mutations in POGLUT1), Spondylocostal Dysostosis 3 (autosomal recessive mutations in LFNG), Adams-Oliver syndrome (autosomal recessive mutations in EOGT), and some cancers (amplification, gain or loss-of-function of POFUT1, Fringe enzymes, POGLUT1, MGAT3). Here we review the characteristics of these diseases and potential molecular mechanisms.

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.

Novel mouse GABA uptake inhibitors with enhanced inhibitory activity toward mGAT3/4 and their effect on pain threshold in mice.

γ-Aminobutyric acid (GABA) uptake transporters are membrane transport proteins that are involved in the pathophysiology of a number of neurological disorders. Some types of chronic pain appear to result from the dysfunction of the GABAergic system. The deficiency of mouse GAT1 transporter (mGAT1) abolishes the nociceptive response, which means that mGAT1 inhibition is an appropriate medical approach to achieve analgesia. The mGAT4 transporter is the second most abundant GAT subtype in the brain; however, its physiological role has not yet been fully understood in the central nervous system. In this study, we examined whether the combination of mGAT1 and mGAT3/mGAT4 inhibition in a single molecule might lead to potentially synergistic effects improving analgesic activity to relieve neuropathic pain. To study this hypothesis, new GABA uptake inhibitors were designed, synthesized, and evaluated in terms of their activity and subtype selectivity for mGAT1-4. Among new functionalized amino acid derivatives of serine and GABA analogs, compounds with preferential mGAT3/4 inhibitory activity were discovered. Two selected hits (19b and 31c) were subjected to in vivo tests. We found a statistically significant antiallodynic activity in the von Frey test in diabetic and oxaliplatin-induced neuropathic pain model. The novel compounds (4-hydroxybutanoic, 4-hydroxypentanoic, and 4-aminobutanoic acid derivatives and serine analogs) provide new insights into the structure-activity relationship of mGAT3/mGAT4 inhibitors and indicate a new direction in the search for potential treatment of neuropathic pain of various origin.

Awakening dormant glycosyltransferases in CHO cells with CRISPRa.

Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.

Bisecting N-Acetylglucosamine Structures Inhibit Hypoxia-Induced Epithelial-Mesenchymal Transition in Breast Cancer Cells.

The epithelial-mesenchymal transition (EMT) process plays a key role in many biological processes, including tissue fibrosis, metastatic diseases, and cancer progression. EMT can be induced by certain factors, notably hypoxia, in the tumor microenvironment. Aberrant levels of certain N-glycans is associated with cancer progression. We used an integrated strategy (mass spectrometry in combination with lectin microarray analysis) to elucidate aberrant glycosylation in a hypoxia-induced EMT model using breast cancer cell lines MCF7 and MDA-MB-231. The model showed reduced levels of bisecting GlcNAc structures, and downregulated expression of the corresponding glycosyltransferase MGAT3. MGAT3 overexpression in MCF7 suppressed cell migration, proliferation, colony formation, expression of EMT markers, and AKT signaling pathway, whereas MGAT3 knockdown (shRNA silencing) had opposite effects. Our findings clearly demonstrate the functional role (and effects of dysregulation) of bisecting GlcNAc structures in hypoxia-induced EMT, and provide a useful basis for further detailed studies of physiological functions of these structures in breast cancer.

Synthesis and biological evaluation of a series of N-alkylated imidazole alkanoic acids as mGAT3 selective GABA uptake inhibitors.

In this paper, we report the synthesis and biological evaluation of a series of 1,5- and 1,4- substituted derivatives of 1H-imidazol-4-ylacetic acid, a series of 1,2-substituted 3-(1H-imidazol-2-yl)propanoic acid and an N-substituted (2E)-3-(1H-imidazol-2-yl)prop-2-enoic acid as new mGAT3 inhibitors. The lipophilic moieties attached to the N-atom of the parent structures were delineated from the 2-[9-(4-methoxyphenyl)-9H-fluoren-9-yl]oxyethyl residue, known from a prototypic mGAT3 inhibitor. For the structure-activity-relationship studies, the spacer between the N-atom of the imidazole ring and the 2-[9-(4-methoxyphenyl)-9H-fluoren-9-yl] moiety was varied in length from three to six atoms, and in nature being either a pure saturated or unsaturated alkyl chain or an alkyl chain containing up to two ether functions. The compounds were characterized for inhibitory potencies at mouse GABA transporter proteins mGAT1-mGAT4. Among the 1,2-substituted compounds, the N-alkylated (2E)-3-(1H-imidazol-2-yl)prop-2-enoic acid 12e containing a C5O spacer exhibits a pIC50 value of 5.13 ± 0.04 at mGAT3, but is devoid of significant selectivity for this GABA transporter. However, the inhibitory potency displayed by 12e at mGAT3 nominally surpasses that of SNAP-5294 reported as the most potent inhibitor of mGAT3 so far.

CSF N-glycoproteomics for early diagnosis in Alzheimer's disease.

This work aims at exploring the human CSF (Cerebrospinal fluid) N-glycome by MALDI MS techniques, in order to assess specific glycosylation pattern(s) in patients with Alzheimer's disease (n:24) and in subjects with mild cognitive impairment (MCI) (n:11), these last as potential AD patients at a pre-dementia stage. For comparison, 21 healthy controls were studied. We identified a group of AD and MCI subjects (about 40-50% of the studied sample) showing significant alteration of CSF N-glycome profiling, consisting of a decrease in the overall sialylation degree and an increase in species bearing bisecting GlcNAc. Noteworthy, all the MCI patients that converted to AD within the clinical follow-up, had an abnormal CSF glycosylation profile. Based on the studied cohort, CSF glycosylation changes may occur before an AD clinical onset. Previous studies specifically focused on the key role of glycosyltransferase GnT-III on AD-pathogenesis, addressing the patho-mechanism to specific sugar modification of BACE-1 glycoprotein with bisecting GlcNAc. Our patients addressed protein N-glycosylation changes at an early phase of the whole biomolecular misregulation on AD, pointing to CSF N-glycome analyses as promising tool to enhance early detection of AD and also suggesting alternative therapeutics target molecules, such as specific glyco-enzymes.

An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease.

The ß-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-ß (Aß) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of Aß-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aß plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics.

Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer.

Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3(-/-)/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3(-/-)/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in ∼20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer.

The bisecting GlcNAc on N-glycans inhibits growth factor signaling and retards mammary tumor progression.

The branching of complex N-glycans attached to growth factor receptors promotes tumor progression by prolonging growth factor signaling. The addition of the bisecting GlcNAc to complex N-glycans by Mgat3 has varying effects on cell adhesion, cell migration, and hepatoma formation. Here, we show that Chinese hamster ovary cells expressing Mgat3 and the polyoma middle T (PyMT) antigen have reduced cell proliferation and growth factor signaling dependent on a galectin lattice. The Mgat3 gene is not expressed in virgin mammary gland but is upregulated during lactation and is expressed in mouse mammary tumor virus (MMTV)/PyMT tumors. Mice lacking Mgat3 that cannot transfer the bisecting GlcNAc to N-glycans acquire PyMT-induced mammary tumors more rapidly and have an increased tumor burden, increased migration of tumor cells, and increased early metastasis to lung. Tumors and tumor-derived cells lacking Mgat3 exhibit enhanced signaling through the Ras pathway and reduced amounts of functionally glycosylated alpha-dystroglycan. Constitutive overexpression of an MMTV/Mgat3 transgene inhibits early mammary tumor development and tumor cell migration. Thus, the addition of the bisecting GlcNAc to complex N-glycans of mammary tumor cell glycoprotein receptors is a cell autonomous mechanism serving to retard tumor progression by reducing growth factor signaling.