B3GALT4 remodels the tumor microenvironment through GD2-mediated lipid raft formation and the c-met/AKT/mTOR/IRF-1 axis in neuroblastoma.

BACKGROUND: Beta-1,3-galactosyltransferase-4 (B3GALT4) plays a critical regulatory role in tumor biology. However, the role of B3GALT4 in modulating the tumor microenvironment (TME) of neuroblastoma (NB) remains unknown. METHODS: Public datasets and clinical NB samples were collected to evaluate the expression and clinical significance of GD2 and B3GALT4 in NB patients. CCK-8, colony formation, and transwell assays and experiments in tumor-bearing mouse models were conducted to investigate the function of B3GALT4. Flow cytometry, ELISA, immunohistochemistry, immunofluorescence, western blotting, and chemotaxis assays were conducted to ascertain the immunomodulatory mechanism of B3GALT4. The combined therapeutic effect of the lipid raft inhibitor MßCD and anti-GD2 mAb was validated in a murine model of NB. RESULTS: GD2 was overexpressed in NB tissues and high expression of GD2 was associated with poor prognosis in NB patients. B3GALT4 was downregulated in NB tissues, and low expression of B3GALT4 indicated poor prognosis in NB patients. Silencing B3GALT4 significantly enhanced tumor progression both in vitro and in vivo. Meanwhile, the overexpression of B3GALT4 increased the recruitment of CD8+ T lymphocytes via the chemokines CXCL9 and CXCL10. Additionally, B3GALT4 regulated NB-cell GD2 expression and lipid raft formation. Mechanistically, B3GALT4 regulated the expression of CXCL9 and CXCL10 via the c-Met signaling in the lipid rafts and the downstream AKT/mTOR/IRF-1 pathway. The lipid raft inhibitor, MßCD, attenuated B3GALT4 deficiency-induced tumor progression and immune evasion. Last, MßCD combined with anti-GD2 mAb treatment significantly enhanced the antitumor effect and the infiltration of CD8+ T cells. CONCLUSIONS: Upregulation of B3GALT4 promotes the secretion of CXCL9 and CXCL10 to recruit CD8+ T lymphocytes via the GD2-mediated lipid rafts and the c-Met/AKT/mTOR/IRF-1 pathway. Moreover, lipid raft inhibitors may enhance the efficacy of anti-GD2 immunotherapy for NB.

Intranasal administration of ß-1, 3-galactosyltransferase 2 confers neuroprotection against ischemic stroke by likely inhibiting oxidative stress and NLRP3 inflammasome activation.

Ischemic stroke is one of the major causes of morbidity and mortality. The ß-1, 3-galactosyltransferase 2 (B3galt2), a member of ß-1, 3-galactosyltransferase family, is playing a vital role in the pathological process of cerebral ischemic injury, but its underlying mechanisms remain unclear. In the present study, we examined the involvement of oxidative stress and NLRP3 inflammasome activation in the neuroprotective effect of B3galt2. Cerebral ischemia/reperfusion (I/R) injury was simulated in a mouse middle cerebral artery occlusion (MCAO) model. Recombinant human B3galt2 (rh-B3galt2) was administered intranasally 1 h post MCAO, and TGF-ß1-siRNA was administered intracerebroventricularly 24 h before MCAO. Outcome measures included brain infarct volume, neurological function, blood-brain barrier (BBB) permeability, neuronal apoptosis, oxidative stress, and the inflammatory response. First, we found that rh-B3galt2 significantly alleviated brain infarct volume and BBB permeability, improved neurological function, and attenuated I/R-induced neuron apoptosis and oxidative stress. Furthermore, rh-B3galt2 attenuated pro-inflammatory cytokines, NF-κB, IL-6, TNF-α, and IL-1ß, and inhibited NLRP3 inflammasome activation. Finally, inhibition of TGF-ß1 by TGF-ß1-siRNA abolished the anti-oxidative and anti-inflammatory effects of rh-B3galt2 in mice after I/R. Collectively, our study demonstrated that rh-B3galt2 exerts neuroprotective effects by regulating cerebral ischemia-induced oxidative stress and NLRP3 inflammasome, which is mainly dependent on the heightening of the TGF-ß1 pathway. Thus, B3galt2 might be considered a new therapeutic target for ischemic stroke treatment.

Challenge to the suppression of tumor growth by the ß4-galactosyltransferase genes.

It has been well established that structural changes in glycans attached to proteins and lipids are associated with malignant transformation of cells. We focused on galactose residues among the sugars since they are involved in the galectin-mediated biology, and many carbohydrate antigens are frequently expressed on this sugar. We found changes in the expression of the ß4-galactosyltransferase (ß4GalT) 2 and 5 genes in cancer cells: decreased expression of the ß4GalT2 gene and increased expression of the ß4GalT5 gene. The growth of mouse melanoma cells showing enhanced expression of the ß4GalT2 gene or reduced expression of the ß4GalT5 gene is inhibited remarkably in syngeneic mice. Tumor growth inhibition is probably caused by the induction of apoptosis, inhibition of angiogenesis, and/or reduced MAPK signals. Direct transduction of human ß4GalT2 cDNA together with the adenovirus vector into human hepatocellular carcinoma cells grown in SCID mice results in marked growth retardation of the tumors. ß4GalT gene-transfer appears to be a potential tool for cancer therapy.

Autologous tumor vaccines processed to express alpha-gal epitopes: a practical approach to immunotherapy in cancer.

This review describes a method by which the human natural anti-Gal antibody can be exploited as an endogenous adjuvant for targeting autologous tumor vaccines to antigen-presenting cells (APCs). Tumor cells remaining in the patient after completion of surgery, radiation, and chemotherapy are the cause of tumor relapse. These residual tumor cells can not be detected by imaging, but their destruction may be feasible by active immunotherapy. Since specific tumor-associated antigens (TAAs) have not been identified for the majority of cancers, irradiated autologous tumor vaccines have been considered as an immunotherapy treatment that may elicit an immune response against the residual tumor cells expressing TAAs. However, tumor cells evolve in cancer patients in a stealthy way, i.e., they are not detected by APCs, even in the form of vaccine. Effective targeting of tumor vaccines for uptake by APCs is a prerequisite for eliciting an effective immune response which requires transport of the vaccine by APCs from the vaccination site to the draining lymph nodes. In the lymph nodes, the APCs transporting the vaccine process and present peptides, including the autologous TAA peptides for activation of the tumor-specific T cells. The required targeting of vaccines to APCs is feasible in humans by the use of anti-Gal. This antibody interacts specifically with the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) and is the only known natural IgG antibody to be present in large amounts in all humans who are not severely immunocompromised. The alpha-gal epitope can be synthesized on any type of human tumor cell by the use of recombinant alpha1,3galactosyltransferase (alpha1,3GT). Solid tumors obtained from surgery are homogenized and their membranes subjected to alpha-gal epitope synthesis. Similarly, alpha-gal epitopes can be synthesized on intact tumor cells from hematological malignancies. Administration of irradiated autologous tumor vaccines processed to express alpha-gal epitopes results in in situ opsonization of the vaccinating cells or cell membranes due to anti-Gal binding to these epitopes. The bound antibody serves to target the autologous tumor vaccine to APCs because the Fc portion of the antibody interacts with Fcgamma receptors on APCs. Since patients receive their own TAAs, the vaccine is customized for autologous TAAs in the individual patient. The repeated vaccination with such autologous tumor vaccines provides the immune system of each patient with an additional opportunity to be effectively activated by the autologous TAAs. In some of the immunized patients this activation may be potent enough to induce an immune-mediated eradication of the residual tumor cells expressing these TAAs.

Inhibition of primary and metastatic tumor growth in mice by cancer-associated galactosyltransferase acceptor.

The antitumor activity of a glycopeptide purified from human malignant effusion, termed cancer-associated galactosyltransferase acceptor (CAGA), was assessed in BALB/c mice bearing primary and metastatic tumors. Initial studies with the fast-growing KA31 and slow-growing KB521 Kirsten sarcoma-transformed mouse fibroblast cell lines confirmed their tumorigenicity and metastatic potential. Inoculation of 1 X 10(5) KA31 cells s.c. resulted in palpable tumor formation in recipient animals within 14 days and death within 42 days from primary tumor growth (mean survival, 26 days; total survival, 0%). Inoculation of the slower-growing KB521 resulted in tumor formation in 85% of recipients, and tumor-bearing animals succumbed within 56 days after primary inoculation (mean survival, 48 days; total survival, 15%). Administration of CAGA by i.p. injection as a single dose or series of five daily doses (each 50 micrograms) inhibited primary tumor growth by 35 to 68% in animals receiving KA31 cells and by 25 to 70% in animals receiving KB521 cells. CAGA increased mean survival 50% from 26 to 38 days and total survival from 0 to 27% in animals bearing KA31-derived primary tumors. In animals bearing KB521-derived tumors, CAGA increased mean survival from 48 to 90 days and total survival from 15 to 50%. Similarly, CAGA was also found to significantly inhibit formation of pulmonary metastases in animals after excision of primary tumors. CAGA administration reduced death from metastatic deposits by 55 to 66% in animals initially inoculated with the KA31 cell line and by 58 to 90% in animals initially bearing primary tumors derived from the KB521 line. There was a corresponding decrease in the number of metastatic deposits per lung after administration of CAGA. Thus, CAGA appears to have potential antitumor activity against tumors with a range of growth rates and appears to inhibit both primary and metastatic tumor growth.