Overexpression of GalNAc-transferase GalNAc-T3 Promotes Pancreatic Cancer Cell Growth

O-linked glycans of secreted and membrane bound proteins play an important role in the pathogenesis of pancreatic cancer by modulating immune responses, inflammation, and tumorigenesis. A critical aspect of O-glycosylation, the position at which proteins are glycosylated with N-acetyl-galactosamine on serine and threonine residues, is regulated by the substrate specificity of UDP-GalNAc: polypeptide N-acetylgalactosaminyl-transferases (GalNAc-Ts). Thus, GalNAc-Ts regulate the first committed step in O-glycosylated protein biosynthesis, determine sites of O-glycosylation on proteins, and are important for understanding normal and carcinoma-associated O-glycosylation. We have found that one of these enzymes, GalNAc-T3, is overexpressed in human pancreatic cancer tissues, and suppression of GalNAc-T3 significantly attenuates growth of pancreatic cancer cells in vitro and in vivo. In addition, suppression of GalNAc-T3 induces apoptosis of pancreatic cancer cells. Our results indicate that GalNAc-T3 is likely to be involved in pancreatic carcinogenesis. Modification of cellular glycosylation occurs in nearly all types of cancer as a result of alterations in the expression levels of glycosyltransferases. We report guanine nucleotide binding protein, alpha transducing activity polypeptide 1 (GNAT1) as a possible substrate protein of GalNAc-T3. GalNAc-T3 is associated with O-glycosylation of GNAT1, and affects the subcellular distribution of GNAT1. Knocking down endogenous GNAT1 significantly suppresses the growth/survival of PDAC cells. Our results imply that GalNAc-T3 contributes to the function of O-glycosylated proteins and thereby affects the growth and survival of pancreatic cancer cells. Thus, substrate proteins of GalNAc-T3 should serve as important therapeutic targets for pancreatic cancers

Overexpression of GalNAc-transferase GalNAc-T3 Promotes Pancreatic Cancer Cell Growth

O-linked glycans of secreted and membrane bound proteins play an important role in the pathogenesis of pancreatic cancer by modulating immune responses, inflammation, and tumorigenesis. A critical aspect of O-glycosylation, the position at which proteins are glycosylated with N-acetyl-galactosamine on serine and threonine residues, is regulated by the substrate specificity of UDP-GalNAc: polypeptide N-acetylgalactosaminyl-transferases (GalNAc-Ts). Thus, GalNAc-Ts regulate the first committed step in O-glycosylated protein biosynthesis, determine sites of O-glycosylation on proteins, and are important for understanding normal and carcinoma-associated O-glycosylation. We have found that one of these enzymes, GalNAc-T3, is overexpressed in human pancreatic cancer tissues, and suppression of GalNAc-T3 significantly attenuates growth of pancreatic cancer cells in vitro and in vivo. In addition, suppression of GalNAc-T3 induces apoptosis of pancreatic cancer cells. Our results indicate that GalNAc-T3 is likely to be involved in pancreatic carcinogenesis. Modification of cellular glycosylation occurs in nearly all types of cancer as a result of alterations in the expression levels of glycosyltransferases. We report guanine nucleotide binding protein, alpha transducing activity polypeptide 1 (GNAT1) as a possible substrate protein of GalNAc-T3. GalNAc-T3 is associated with O-glycosylation of GNAT1, and affects the subcellular distribution of GNAT1. Knocking down endogenous GNAT1 significantly suppresses the growth/survival of PDAC cells. Our results imply that GalNAc-T3 contributes to the function of O-glycosylated proteins and thereby affects the growth and survival of pancreatic cancer cells. Thus, substrate proteins of GalNAc-T3 should serve as important therapeutic targets for pancreatic cancers

Proximity proteomics identifies cancer cell membrane cis‐molecular complex as a potential cancer target

Cancer-specific antigens expressed in the cell membrane have been used as targets for several molecular targeted strategies in the last 20 years with remarkable success. To develop more effective cancer treatments, novel targets and strategies for targeted therapies are needed. Here, we examined the cancer cell membrane-resident "cis-bimolecular complex" as a possible cancer target (cis-bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT were detected using a previously developed method termed the enzyme-mediated activation of radical source (EMARS), to label the components proximal to a given cell membrane molecule. EMARS analysis identified some BiCAT, such as close homolog of L1 (CHL1), fibroblast growth factor 3 (FGFR3) and alpha2 integrin, which are commonly expressed in mouse primary lung cancer cells and human lung squamous cell carcinoma cells. Analysis of cancer specimens from 55 lung cancer patients revealed that CHL1 and alpha2 integrin were highly co-expressed in almost all cancer tissues compared with normal lung tissues. As an example of BiCAT application, in vitro simulation of effective drug combinations used for multiple drug treatment strategies was performed using reagents targeted to BiCAT molecules. The combination treatment based on BiCAT information moderately suppressed cancer cell proliferation compared with single administration, suggesting that the information about BiCAT in cancer cells is useful for the appropriate selection of the combination among molecular targeted reagents. Thus, BiCAT has the potential to contribute to several molecular targeted strategies in future

Tumor-dependent secretion of close homolog of L1 results in elevation of its circulating level in mouse model for human lung tumor

Close homolog of L1 (CHL1) and its truncated form mainly play crucial roles in mouse brain development and neural functions. Herein, we newly identified that truncated form of CHL1 is produced and released from lung tumor tissue in a mouse model expressing human EML4-ALK fusion gene. Both western blot and direct ELISA analysis revealed that mouse CHL1 level in serum (including serum extracellular vesicles) was significantly elevated in EML4-ALK transgenic mice. The correlation between the tumor size and the amount of CHL1 secretion could be examined in this study, and showed a significant positive correlation in a tumor size-dependent manner. Considering these results, the measurement of circulating CHL1 level may contribute to assess a tumor progression in human lung tumor patients

A Myelin Galactolipid, Sulfatide, Is Essential for Maintenance of Ion Channels on Myelinated Axon But Not Essential for Initial Cluster Formation

Myelinated axons are divided into four distinct regions: the node of Ranvier, paranode, juxtaparanode, and internode, each of which is characterized by a specific set of axonal proteins. Voltage-gated N

Identification of Cell-Surface Molecular Interactions under Living Conditions by Using the Enzyme-Mediated Activation of Radical Sources (EMARS) Method

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Expressed glycosylphosphatidylinositol-anchored horseradish peroxidase identifies co-clustering molecules in individual lipid raft domains.

Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains