Beta-1,4-galactosyltransferase-3 deficiency suppresses the growth of immunogenic tumors in mice.

Background: Beta-1,4-galactosyltransferase-3 (B4GALT3) belongs to the family of beta-1,4-galactosyltransferases (B4GALTs) and is responsible for the transfer of UDP-galactose to terminal N-acetylglucosamine. B4GALT3 is differentially expressed in tumors and adjacent normal tissues, and is correlated with clinical prognosis in several cancers, including neuroblastoma, cervical cancer, and bladder cancer. However, the exact role of B4GALT3 in the tumor immune microenvironment (TIME) remains unclear. Here, we aimed to elucidate the function of B4GALT3 in the TIME. Methods: To study the functions of B4GALT3 in cancer immunity, either weakly or strongly immunogenic tumor cells were subcutaneously transplanted into wild-type (WT) and B4galt3 knockout (KO) mice. Bone marrow transplantation and CD8+ T cell depletion experiments were conducted to elucidate the role of immune cells in suppressing tumor growth in B4galt3 KO mice. The cell types and gene expression in the tumor region and infiltrating CD8+ T cells were analyzed using flow cytometry and RNA sequencing. N-glycosylated proteins from WT and B4galt3 KO mice were compared using the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based glycoproteomic approach. Results: B4galt3 KO mice exhibited suppressed growth of strongly immunogenic tumors with a notable increase in CD8+ T cell infiltration within tumors. Notably, B4galt3 deficiency led to changes in N-glycan modification of several proteins, including integrin alpha L (ITGAL), involved in T cell activity and proliferation. In vitro experiments suggested that B4galt3 KO CD8+ T cells were more susceptible to activation and displayed increased downstream phosphorylation of FAK linked to ITGAL. Conclusion: Our study demonstrates that B4galt3 deficiency can potentially boost anti-tumor immune responses, largely through enhancing the influx of CD8+ T cells. B4GALT3 might be suppressing cancer immunity by synthesizing the glycan structure of molecules on the CD8+ T cell surface, as evidenced by the changes in the glycan structure of ITGAL in immune cells. Importantly, B4galt3 KO mice showed no adverse effects on growth, development, or reproduction, underscoring the potential of B4GALT3 as a promising and safe therapeutic target for cancer treatment.

A degron system targeting endogenous PD-1 inhibits the growth of tumor cells in mice.

Recently, targeted protein degradation systems have been developed using the ubiquitin-proteasome system. Here, we established Programmed cell death-1 (PD-1) knockdown mice as a model system for subjecting endogenous mouse proteins to the small molecule-assisted shutoff (SMASh) degron system. SMASh degron-tagged PD-1-mCherry in Jurkat cells and CD3+ splenocytes were degraded by the NS3/4A protease inhibitors, asunaprevir (ASV) or grazoprevir (GRV). Growth of MC-38 colon adenocarcinoma cells injected in Pdcd1-mCherry-SMASh homozygous knock-in (KI) mice was repressed by ASV or GRV. Moreover, growth of MC-38 cells was suppressed in wild-type mice transplanted with KI bone marrow cells after GRV treatment. This is the first study to use a degron tag targeting an endogenous mouse protein in vivo. Our experimental system using the SMASh degron may be employed for treating diseases and characterizing the cellular functions of essential proteins.

Involvement of AMP-activated Protein Kinase α/Nuclear Factor (Erythroid-derived 2) Like 2-iniatived Signaling Pathway in Cytoprotective Effects of Wasabi 6-(Methylsulfinyl) Hexyl Isothiocyanate.

6-(Methylsulfinyl) hexyl isothiocyanate (6-MSITC) is an active ingredient present in Wasabi, which is a popular pungent spice used in Japanese cuisine. Our previous studies suggested that the primary antioxidant activity of 6-MSITC may link to other biological activity. This study aimed to clarify how the antioxidant activity of 6-MSITC contributes to preventing overloaded lipid stress in hepatic cell model. HepG2 cells were treated with 6-MSITC at defined concentrations and times in normal medium or in combined fatty acids (CFA) medium, and the targeted proteins were detected by Western blotting. The kinetic data revealed that 6-MSITC activated AMP-activated protein kinase α (AMPKα) and nuclear factor (erythroid-derived 2) like 2 (Nrf2), and then enhanced the protein expression of Forkhead box protein O1 (FOXO1) and Sirtuin1 as well as that of the Nrf2 target proteins, NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase (HO-1). Furthermore, lipid metabolic stress was mimicked in HepG2 cells by overloading CFA. 6-MSITC significantly alleviated CFA-induced formation of thiobarbituric acid reactive substances and fat accumulation. Signaling analysis data revealed that 6-MSITC enhanced phosphorylation of AMPKα, upregulated the expression of Nrf2, NQO1, heme oxygenase 1, FOXO1, and Siruin1, and downregulated the expression of PPARα. Taken together, our results suggested that the AMPKα/Nrf2-mediated signaling pathways might be involved in the cytoprotective effects of Wasabi 6-MSITC against metabolic lipid stress.