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Summary A short form of cellular FLICE-inhibitory protein encoded by CFLARs promotes necroptosis. Although necroptosis is involved in various pathological conditions, the detailed mechanisms are not fully understood. Here we generated transgenic mice wherein CFLARs was integrated onto the X chromosome. All male CFLARs Tg mice died perinatally due to severe ileitis. Although necroptosis was observed in various tissues of CFLARs Tg mice, large numbers of intestinal epithelial cells (IECs) died by apoptosis. Deletion of Ripk3 or Mlkl, essential genes of necroptosis, prevented both necroptosis and apoptosis, and rescued lethality of CFLARs Tg mice. Type 3 innate lymphoid cells (ILC3s) were activated and recruited to the small intestine along with upregulation of interleukin-22 (Il22) in CFLARs Tg mice. Deletion of ILC3s or Il22 rescued lethality of CFLARs Tg mice by preventing apoptosis, but not necroptosis of IECs. Together, necroptosis-dependent activation of ILC3s induces lethal ileitis in an IL-22-dependent manner., Graphical Abstract, Highlights • CFLARs Tg mice develop severe ileitis in utero • Intestinal epithelial cells die by apoptosis and necroptosis in CFLARs Tg mice • Blockade of necroptosis rescues lethality of CFLARs Tg mice • Necroptosis activates type 3 innate lymphoid cells, resulting in severe ileitis, Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology

Receptor interacting protein kinase 1 (RIPK1) is a cytosolic multidomain protein that controls cell life and death. While RIPK1 promotes cell death through its kinase activity, it also functions as a scaffold protein to promote cell survival by inhibiting FADD-caspase 8-dependent apoptosis and RIPK3-MLKL-dependent necroptosis. This pro-survival function is highlighted by excess cell death and a perinatal lethality in Ripk1−/− mice. Recently, loss of function mutation of RIPK1 was found in patients with immunodeficiency and inflammatory bowel diseases. Hematopoietic stem cell transplantation restored not only immunodeficiency but also intestinal inflammatory pathology, indicating that RIPK1 in hematopoietic cells is critical to maintain intestinal immune homeostasis. Here, we generated dendritic cell (DC)-specific Ripk1−/− mice in a genetic background with loss of RIPK1 kinase activity and found that the mice developed spontaneous colonic inflammation characterized by increased neutrophil infiltration. In addition, these mice were highly resistant to injury-induced colitis. The increased neutrophil infiltration in the colon and the resistance to colitis were restored by dual inactivation of RIPK3 and FADD, but not by inhibition of RIPK3, MLKL, or ZBP1 alone. Altogether, these results reveal a scaffold activity-dependent role of RIPK1 in protecting colonic DCs from apoptotic insults and maintenance of colonic immune homeostasis.

We previously found that the serum level of fucosylated haptoglobin (Fuc-Hpt) was significantly increased in pancreatic cancer patients. To delineate the mechanism underlying this increase and develop a simple detection method, we set out to generate a monoclonal antibody (mAb) specific for Fuc-Hpt. After multiple screenings by enzyme-linked immunosorbent assay (ELISA), a 10-7G mAb was identified as being highly specific for Fuc-Hpt generated in a cell line as well as for Hpt derived from a pancreatic cancer patient. As a result from affinity chromatography with 10-7G mAb, followed by lectin blot and mass spectrometry analyses, it was found that 10-7G mAb predominantly recognized both Fuc-Hpt and prohaptoglobin (proHpt), which was also fucosylated. In immunohistochemical analyses, hepatocytes surrounding metastasized cancer cells were stained by the 10-7G mAb, but neither the original cancer cells themselves nor normal hepatocytes exhibited positive staining, suggesting that metastasized cancer cells promote Fuc-Hpt production in adjacent hepatocytes. Serum level of Fuc-Hpt determined with newly developed ELISA system using the 10-7G mAb, was increased in patients of pancreatic and colorectal cancer. Interestingly, dramatic increases in Fuc-Hpt levels were observed at the stage IV of colorectal cancer. These results indicate that the 10-7G mAb developed is a promising antibody which recognizes Fuc-Hpt and could be a useful diagnostic tool for detecting liver metastasis of cancer., This study was performed as a research program of the Project for Development of Innovative Research on Cancer Therapeutics (P-Direct), Ministry of Education, Culture, Sports, Science and Technology of Japan and was supported by JSPS KAKENHI Grant Number JP16H05226.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) fixes atmospheric carbon dioxide into bioavailable sugar molecules. It is also well known that a kinetic isotope effect (KIE; CO2 carbon atoms) accompanies the carboxylation process. To describe the reaction and the KIE α, two different types of molecular dynamics (MD) simulations (ab initio MD and classical MD) have been performed with an Own N-layered Integrated molecular Orbitals and molecular Mechanics (ONIOM)-hybrid model. A channel structure for CO2 transport has been observed during the MD simulation in RubisCO, and assuming the reaction path from the inlet to the product through the coordinate complex with Mg2+ , simulations have been performed on several molecular configuration models fixing several distances between CO2 and ribulose-1,5-bisphosphate along the channel. Free energy analysis and diffusion coefficient analysis have been evaluated for different phases of the process. It is confirmed that the isotopic fractionation effect for CO2 containing either 13 C or 12 C would appear through the transiting path in the channel structure identified in RubisCO. The estimated isotope fractionation constant was quite close to the experimental value.

Recent advances have identified a signaling cascade involving receptor interacting protein kinase 1 (RIPK1), RIPK3 and the pseudokinase mixed lineage kinase domain-like (MLKL) that is crucial for induction of necroptosis, a non-apoptotic form of cell death. RIPK1–RIPK3–MLKL-mediated necroptosis has been attributed to cause many inflammatory diseases through the release of cellular damage-associated molecular patterns (DAMPs). In addition to necroptosis, emerging evidence suggests that these necroptosis signal adaptors can also facilitate inflammation independent of cell death. In particular, the RIP kinases can drive NF-κB and inflammasome activation independent of cell death. In this review, we will discuss recent discoveries that led to this realization and present arguments why cell death-independent signaling by the RIP kinases may have a more important role in inflammation than necroptosis.

Receptor-interacting protein kinase 3 (RIPK3) is a serine/threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.

Necroptosis is an emerging mode of programmed cell death that is defined at the molecular level through involvement of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL). In contrast to apoptosis, phosphorylation is a key event in necroptosis. Necroptosis can be triggered in response to various cell-surface receptors, such as death receptors in the tumor necrosis factor receptor family, Toll-like receptors, and interferon receptors. Because apoptotic machinery is often impaired in cancer cells, necroptosis has garnered attention as a promising strategy to treat apoptosis-resistant cancer. Moreover, since necroptosis leads to release of intracellular immunogenic contents and is therefore highly immunogenic, it has the potential to promote robust antitumor adaptive immune response. In this chapter, we describe current understanding of the role of necroptosis in cancer progression and discuss strategies through which we can harness the power of necroptosis in cancer therapy.

The cytokine IL-1β is intimately linked to many pathological inflammatory conditions. Mature IL-1β secretion requires cleavage by the inflammasome. Recent evidence indicates that many cell death signal adaptors have regulatory roles in inflammasome activity. These include the apoptosis inducers FADD and caspase 8, and the necroptosis kinases receptor interacting protein kinase 1 (RIPK1) and RIPK3. PGAM5 is a mitochondrial phosphatase that has been reported to function downstream of RIPK3 to promote necroptosis and IL-1β secretion. To interrogate the biological function of PGAM5, we generated Pgam5−/− mice. We found that Pgam5−/− mice were smaller compared with wild type littermates, and male Pgam5−/− mice were born at sub-Mendelian ratio. Despite these growth and survival defects, Pgam5−/− cells responded normally to multiple inducers of apoptosis and necroptosis. Rather, we found that PGAM5 is critical for IL-1β secretion in response to NLRP3 and AIM2 inflammasome agonists. Moreover, vesicular stomatosis virus–induced IL-1β secretion was impaired in Pgam5−/− bone marrow–derived macrophages, but not in Ripk3−/− bone marrow–derived dendritic cells, indicating that PGAM5 functions independent of RIPK3 to promote inflammasome activation. Mechanistically, PGAM5 promotes ASC polymerization, maintenance of mitochondrial integrity, and optimal reactive oxygen species production in response to inflammasome signals. Hence PGAM5 is a novel regulator of inflammasome and caspase 1 activity that functions independently of RIPK3. Fil: Moriwaki, Kenta. University of Massachussets; Estados Unidos Fil: Farias Luz, Nivea. Universidade Federal da Bahia; Brasil. University of Massachussets; Estados Unidos Fil: Balaji, Sakthi. University of Massachussets; Estados Unidos Fil: de Rosa, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: O'Donnell, Carey L.. University of Massachussets; Estados Unidos Fil: Gough, Peter J.. Glaxo Smith Kline; Estados Unidos Fil: Bertin, John. Glaxo Smith Kline; Estados Unidos Fil: Welsh, Raymond M.. University of Massachussets; Estados Unidos Fil: Chan, Francis Ka Ming. University of Massachussets; Estados Unidos

Caspase 8, the initiator caspase for death receptor–induced apoptosis, functions as a negative regulator of receptor interacting protein kinase 3 (RIPK3), an essential factor for TNF-, TLR3-, and TLR4-induced necroptosis. In certain situations, caspase 8 can also participate in pro–IL-1β processing. However, the biochemical complex that mediates caspase 8–mediated processing is not defined. In this study, we show that RIPK3 is crucial for caspase 1– and caspase 8–mediated pro–IL-1β and pro–IL-18 processing in bone marrow–derived dendritic cells (BMDCs) in response to LPS stimulation. Caspase 8–mediated pro–IL-1β processing requires intact RIPK1, RIPK3, TRIF, and FADD. In response to LPS, a complex that contains RIPK1, RIPK3, FADD, and caspase 8 is formed. Surprisingly, RIPK3-specific kinase inhibitors strongly enhanced caspase 8 activation and pro–IL-1β processing in LPS-stimulated BMDCs. However, studies in BMDCs expressing the kinase-inactive RIPK3-K51A mutant or RIPK1-K45A mutant showed that the kinase activity of neither RIPK1 nor RIPK3 is required for LPS-induced caspase 8 activation and IL-1β secretion. Hence, RIPK3 is an unexpected positive regulator of caspase 8 activity that promotes IL-1β maturation in BMDCs.

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

SummaryProgrammed necrosis or necroptosis is an inflammatory form of cell death that critically requires the receptor-interacting protein kinase 3 (RIPK3). Here we showed that RIPK3 controls a separate, necrosis-independent pathway of inflammation by regulating cytokine expression in dendritic cells (DCs). Ripk3−/− bone-marrow-derived dendritic cells (BMDCs) were highly defective in lipopolysaccharide (LPS)-induced expression of inflammatory cytokines. These effects were caused by impaired NF-κB subunit RelB and p50 activation and by impaired caspase 1-mediated processing of interleukin-1β (IL-1β). This DC-specific function of RIPK3 was critical for injury-induced inflammation and tissue repair in response to dextran sodium sulfate (DSS). Ripk3−/− mice exhibited an impaired axis of injury-induced IL-1β, IL-23, and IL-22 cytokine cascade, which was partially corrected by adoptive transfer of wild-type DCs, but not Ripk3−/− DCs. These results reveal an unexpected function of RIPK3 in NF-κB activation, DC biology, innate inflammatory-cytokine expression, and injury-induced tissue repair.

It is now widely accepted that some forms of necrosis are controlled by a dedicated signaling pathway triggered by various cell surface and intracellular receptors. This regulated form of necrosis is mediated by the kinase activity of receptor-interacting protein kinase 1 (RIP1/RIPK1) and/or RIP3/RIPK3. A number of studies using the RIP1 kinase inhibitor Necrostatin-1 (Nec-1) and its derivatives, or RIP3-deficient mice demonstrated that RIP1 and RIP3 are involved in various infectious and sterile inflammatory diseases. As a consequence, these specific phenotypes were construed to depend on necrosis. However, emerging evidence indicates that the RIP1 kinase activity and RIP3 can also control apoptosis and inflammatory cytokine production independent of necrosis. Therefore, we may need to re-interpret conclusions drawn based on loss of RIP1 or RIP3 functions in in vivo models. We propose that studies of RIP1 and RIP3 in different inflammatory responses need to consider cell death-dependent and independent mechanisms of the RIP kinases.

BIG1, an activator protein of the small GTPase, Arf, and encoded by the Arfgef1 gene, is one of candidate genes for epileptic encephalopathy. To know the involvement of BIG1 in epileptic encephalopathy, we analyzed BIG1-deficient mice and found that BIG1 regulates neurite outgrowth and brain development in vitro and in vivo. The loss of BIG1 decreased the size of the neocortex and hippocampus. In BIG1-deficient mice, the neuronal progenitor cells (NPCs) and the interneurons were unaffected. However, Tbr1+ and Ctip2+ deep layer (DL) neurons showed spatial-temporal dependent apoptosis. This apoptosis gradually progressed from the piriform cortex (PIR), peaked in the neocortex, and then progressed into the hippocampus from embryonic day 13.5 (E13.5) to E17.5. The upper layer (UL) and DL order in the neocortex was maintained in BIG1-deficient mice, but the excitatory neurons tended to accumulate before their destination layers. Further pulse-chase migration assay showed that the migration defect was non-cell autonomous and secondary to the progression of apoptosis into the BIG1-deficient neocortex after E15.5. In BIG1-deficient mice, we observed an ectopic projection of corticothalamic axons from the primary somatosensory cortex (S1) into the dorsal lateral geniculate nucleus (dLGN). The thalamocortical axons were unable to cross the diencephalon-telencephalon boundary (DTB). In vitro, BIG1-deficient neurons showed a delay in neuronal polarization. BIG1-deficient neurons were also hypersensitive to low dose glutamate (5 μM), and died via apoptosis. This study showed the role of BIG1 in the survival of DL neurons in developing embryonic brain and in the generation of neuronal polarity.

Receptor interacting protein kinase 3 (RIPK3) is an essential serine/threonine kinase for necroptosis, a type of regulated necrosis. A variety of stimuli can cause RIPK3 activation through phosphorylation. Activated RIPK3 in turn phosphorylates and activates the downstream necroptosis executioner mixed lineage kinase domain-like (MLKL). Necroptosis is a highly inflammatory type of cell death because of the release of intracellular immunogenic contents from disrupted plasma membrane. Indeed, RIPK3-deficient mice exhibited reduced inflammation in many inflammatory disease models. These results have been interpreted as evidence that necroptosis is a key driver for RIPK3-induced inflammation. Interestingly, recent studies show that RIPK3 also regulates NF-κB, inflammasome activation, and kinase-independent apoptosis. These studies also reveal that these non-necroptotic functions contribute significantly to disease pathogenesis. In this review, we summarize our current understanding of necroptotic and non-necroptotic functions of RIPK3 and discuss how these effects contribute to RIPK3-mediated inflammation.

Receptor interacting protein kinase 3 (RIPK3) is a crucial inducer of necroptosis. Its activity is controlled by interaction with other signal adaptors through the “RIP homotypic interaction motif’ (RHIM). Recent studies revealed a critical function for RIPK3 in the maintenance of epithelial tissue integrity. In mice with genetic deficiency of the apoptosis adaptors FADD or caspase 8, RIPK3 promotes necroptotic cell death of epithelial cells, leading to excessive and lethal inflammation. In contrast, when FADD and caspase 8 functions are intact, RIPK3 serves as a protector of intestinal epithelial integrity by promoting injury-induced wound repair. In the latter case, RIPK3 promotes optimal cytokine expression by cells of hematopoietic origin. Specifically, bone marrow derived dendritic cells (BMDCs) have an obligate requirement for RIPK3 for optimal secretion of mature IL-1β and other inflammatory cytokines in response to toll-like receptor 4 (TLR4) stimulation. RIPK3 promotes cytokine expression through two complementary mechanisms: NF-κΒ dependent gene transcription and processing of pro-IL-1β. We propose that RIPK3 functions in different cell compartments to mediate inflammation through distinct mechanisms.

SummaryRIP1 and RIP3 kinases are central players in TNF-induced programmed necrosis. Here, we report that the RIP homotypic interaction motifs (RHIMs) of RIP1 and RIP3 mediate the assembly of heterodimeric filamentous structures. The fibrils exhibit classical characteristics of β-amyloids, as shown by Thioflavin T (ThT) and Congo red (CR) binding, circular dichroism, infrared spectroscopy, X-ray diffraction, and solid-state NMR. Structured amyloid cores are mapped in RIP1 and RIP3 that are flanked by regions of mobility. The endogenous RIP1/RIP3 complex isolated from necrotic cells binds ThT, is ultrastable, and has a fibrillar core structure, whereas necrosis is partially inhibited by ThT, CR, and another amyloid dye, HBX. Mutations in the RHIMs of RIP1 and RIP3 that are defective in the interaction compromise cluster formation, kinase activation, and programmed necrosis in vivo. The current study provides insight into the structural changes that occur when RIP kinases are triggered to execute different signaling outcomes and expands the realm of amyloids to complex formation and signaling.

Oligosaccharides, sequences of carbohydrates conjugated to proteins and lipids, are arguably the most abundant and structurally diverse class of molecules. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and inflammation. Recent advances in glycomics have identified several types of glyco-biomarkers containing fucosylation that are linked to certain types of cancer. Fucosylated alpha-fetoprotein (AFP) is widely used in the diagnosis of hepatocellular carcinoma because it is more specific than alpha-fetoprotein. High levels of fucosylated haptoglobin have also been found in sera of patients with various carcinomas. We have recently established a simple lectin-antibody ELISA to measure fucosylated haptoglobin and to investigate its clinical use. Cellular fucosylation is dependent upon fucosyltransferase activity and the level of its donor substrate, guanosine diphosphate (GDP)-fucose. GDP-mannose-4,6-dehydratase (GMDS) is a key enzyme involved in the synthesis of GDP-fucose. Mutations of GMDS found in colon cancer cells induced a malignant phenotype, leading to rapid growth in athymic mice resistant to natural killer cells. This review describes the role of fucosylated haptoglobin as a cancer biomarker, and discusses the possible biological role of fucosylation in cancer development.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through binding to TRAIL receptors, death receptor 4 (DR4), and DR5. TRAIL has potential therapeutic value against cancer because of its selective cytotoxic effects on several transformed cell types. Fucosylation of proteins and lipids on the cell surface is a very important posttranslational modification that is involved in many cellular events. Recently, we found that a deficiency in GDP-mannose-4,6-dehydratase (GMDS) rendered colon cancer cells resistant to TRAIL-induced apoptosis, resulting in tumor development and metastasis by escape from tumor immune surveillance. GMDS is an indispensable regulator of cellular fucosylation. In this study, we investigated the molecular mechanism of inhibition of TRAIL signaling by GMDS deficiency. DR4, but not DR5, was found to be fucosylated; however, GMDS deficiency inhibited both DR4- and DR5-mediated apoptosis despite the absence of fucosylation on DR5. In addition, GMDS deficiency also inhibited CD95-mediated apoptosis but not the intrinsic apoptosis pathway induced by anti-cancer drugs. Binding of TRAIL and CD95 ligand to their cognate receptors primarily leads to formation of a complex comprising the receptor, FADD, and caspase-8, referred to as the death-inducing signaling complex (DISC). GMDS deficiency did not affect formation of the primary DISC or recruitment to and activation of caspase-8 on the DISC. However, formation of secondary FADD-dependent complex II, comprising caspase-8 and cFLIP, was significantly inhibited by GMDS deficiency. These results indicate that GMDS regulates the formation of secondary complex II from the primary DISC independent of direct fucosylation of death receptors.

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the β1,6 branching of N-acetylglucosamine on N-glycans. GnT-V expression is elevated during malignant transformation in various types of cancer. However, the mechanism by which GnT-V promotes cancer progression is unclear. To characterize the biological significance of GnT-V, we established GnT-V transgenic (Tg) mice, in which GnT-V is regulated by a β-actin promoter. No spontaneous cancer was detected in any organs of the GnT-V Tg mice. However, GnT-V expression was up-regulated in GnT-V Tg mouse skin, and cultured keratinocytes derived from these mice showed enhanced migration, which was associated with changes in E-cadherin localization and epithelial-mesenchymal transition (EMT). Further, EMT-associated factors snail, twist, and N-cadherin were up-regulated, and cutaneous wound healing was accelerated in vivo. We further investigated the detailed mechanisms of EMT by assessing EGF signaling and found up-regulated EGF receptor signaling in GnT-V Tg mouse keratinocytes. These findings indicate that GnT-V overexpression promotes EMT and keratinocyte migration in part through enhanced EGF receptor signaling.

Fucosylated alpha-fetoprotein (AFP) is a specific tumor marker for hepatocellular carcinomas (HCC). However, the mechanisms underlying the increase in fucosylated AFP in serum of patients with HCC are largely unknown. Recently, we reported that fucosylation is a possible signal for the secretion of glycoproteins into bile in the liver. This finding might lead to the selective secretion of fucosylated AFP into bile and the selective secretion might be disrupted in hepatocarcinogenesis. In this study, therefore, we analyzed the oligosaccharide structures of glycoproteins in bile and serum of LEC rats, which are a rat model of spontaneous hepatocarcinogenesis. Lectin microarraying showed enhanced binding of 13 lectins to bile, compared with in serum from normal LEC rats, and the binding of these lectins to serum of LEC rats bearing HCC was higher than in normal rats. Structural analyses involving HPLC and mass spectrometry showed that the fucosylation levels of serum glycoproteins were not increased in CH rats but were in HCC rats, although the fucosylation levels of biliary glycoproteins were increased in both CH and HCC rats. These results suggested that the sorting machinery through fucosylation might be disrupted in the liver with HCC.

Background: Recent advanced techniques in glycobiology have produced a number of tumor marker candidates. As a result from the glycomic approach, we found that fucosylated haptoglobin in sera was a possible tumor marker for pancreatic cancer (PC). Although Aleuria aurantia lectin (AAL) blotting can detect fucosylated haptoglobin, it is difficult to quantify fucosylated haptoglobin precisely. To overcome this problem, we developed a fucosylated haptoglobin detection kit as a sandwich enzyme-linked immune sorbent assay (ELISA) using AAL and the Fab portion of anti-haptoglobin antibody. In the present study, we investigated the clinical application of this lectin-antibody ELISA kit to measure fucosylated haptoglobin in PC. Methods: We measured fucosylated haptoglobin in patients with PC with a lectin-antibody ELISA kit. The fucosylated haptoglobin measured with this assay was compared with lectin blotting data, and the discrepancy was analyzed by immunoprecipitation methods. The concentration of fucosylated haptoglobin was investigated with respect to the clinical stage of PC. We also measured fucosylated haptoglobin, using 397 cases of several types of cancers including PC, benign diseases, and normal controls. Results: The sensitivity and specificity for the differential diagnosis of PC from normal controls was 50% and 91%, respectively. The results from lectin-antibody ELISA were significantly correlated with data from previous AAL blotting studies. Positive rates of fucosylated haptoglobin with this method in patients with PC were significantly higher in cases of stage IV compared with other clinical stages. Fucosylated haptoglobin was increased in several types of cancers, in which fucosylated haptoglobin was reported to increase. Conclusions: While certain cases showed a discrepancy in fucosylated haptoglobin concentrations between the lectin-antibody ELISA and conventional lectin blotting, this novel type of lectin-antibody ELISA might be useful for a tumor marker for PC. Clin Chem Lab Med 2010;48:505–12.

Fucosylation is one of the most important oligosaccharide modifications and is involved in cancer and inflammation. Recently, fucosylated haptoglobin was identified as a possible tumor marker for pancreatic cancer. The molecular mechanism underlying increases in fucosylated haptoglobin in sera of patients with pancreatic cancer seems to be complicated. Our previous study [N. Okuyama, Y. Ide, M. Nakano, T. Nakagawa, K. Yamanaka, K. Moriwaki, K. Murata, H. Ohigashi, S. Yokoyama, H. Eguchi, O. Ishikawa, T. Ito, M. Kato, A. Kasahara, S. Kawano, J. Gu, N. Taniguchi, E. Miyoshi, Fucosylated haptoglobin is a novel marker for pancreatic cancer: a detailed analysis of the oligosaccharide structure and a possible mechanism for fucosylation, Int. J. Cancer 118 (11) (2006) 2803-2808] demonstrated that pancreatic cancer cells secrete a factor, which induces the production of haptoglobin in hepatoma cells. In the present study, we found that interleukin 6 (IL6) expressed in pancreatic cancer is a factor that induces the haptoglobin production, using a neutralizing antibody for IL6. Real-time PCR analyses revealed the up-regulation of fucosylation regulatory genes after IL6 treatment, resulting increases in fucosylated haptoglobin being revealed by a lectin ELISA. This pathway could be one of the possible mechanisms underlying increases in haptoglobin in sera of patients with pancreatic cancer.

Most cancers consist of heterogeneous populations of cells with substantial differences in tumorigenicity. Cells that possess self-renewal and tumor-initiating properties are often called cancer stem cells (CSCs). Since CSCs underlie tumor recurrence and metastasis and are resistant to current anti-cancer therapies, novel therapeutic strategies to efficiently target this subset of cells are needed. Aberrant glycosylation is one of the hallmarks of cancer. Many cancer-associated glycans have been reported to be involved in tumor progression and metastasis, and are used as tumor markers. Over the past several years, we have identified characteristic glycans on CSCs by utilizing recent advances in glycoproteomic technologies. In this review, we would like to summarize a series of our recent studies and discuss possible applications of glycomarkers for CSCs.

Cell proliferation and cell death are integral elements in maintaining homeostatic balance in metazoans. Disease pathologies ensue when these processes are disturbed. A plethora of evidence indicates that malfunction of cell death can lead to inflammation, autoimmunity, or immunodeficiency. Programmed necrosis or necroptosis is a form of nonapoptotic cell death driven by the receptor interacting protein kinase 3 (RIPK3) and its substrate, mixed lineage kinase domain-like (MLKL). RIPK3 partners with its upstream adaptors RIPK1, TRIF, or DAI to signal for necroptosis in response to death receptor or Toll-like receptor stimulation, pathogen infection, or sterile cell injury. Necroptosis promotes inflammation through leakage of cellular contents from damaged plasma membranes. Intriguingly, many of the signal adaptors of necroptosis have dual functions in innate immune signaling. This unique signature illustrates the cooperative nature of necroptosis and innate inflammatory signaling pathways in managing cell and organismal stresses from pathogen infection and sterile tissue injury.

Glycans located on the cell surface regulate cell-cell interaction, cell homing, and signal transmission, which are particularly important for communication among cells. Certain cell types contain unique cell surface glycan structures, which have been utilized as markers for characterization. Flow cytometry is a powerful technology that enables the examination of multiple parameters of individual cells (e.g., cell size, internal complexity, and surface marker expression level). In this chapter, we describe a step-by-step procedure on how to detect glycans on the cell surface of live cells by flow cytometer, using lectins.

Programmed necrosis or necroptosis is a type of cell death regulated by a specific signaling pathway. Hence it is different from necrosis induced by physical trauma. Receptor-interacting protein kinase (RIPK) 1 and RIPK3 play central roles in programmed necrosis by forming a pro-necrotic signaling complex termed the necrosome. Genetic evidence indicates that in concert with the pro-apoptotic molecules FADD and caspase-8, RIPK1 and RIPK3 regulate embryonic development, T cell clonal expansion, and immune homeostasis. Programmed necrosis contributes to innate immune host defense against certain viruses. The importance of programmed necrosis as a host defense mechanism is highlighted by discovery of viral inhibitors of necrosis. Emerging evidence suggests that programmed necrosis is also involved in bacterial infections and may even directly regulate inflammatory cytokine expression. In this chapter, we discuss how the RIP kinases contribute to different inflammatory diseases.

The receptor-interacting protein kinase 3 (RIP3/RIPK3) has emerged as a critical regulator of programmed necrosis/necroptosis, an inflammatory form of cell death with important functions in pathogen-induced and sterile inflammation. RIP3 activation is tightly regulated by phosphorylation, ubiquitination, and caspase-mediated cleavage. These post-translational modifications coordinately regulate the assembly of a macromolecular signaling complex termed the necrosome. Recently, several reports indicate that RIP3 can promote inflammation independent of its pronecrotic activity. Here, we review our current understanding of the mechanisms that drive RIP3-dependent necrosis and its role in different inflammatory diseases.

Fucosylation is a crucial oligosaccharide modification in cancer. The known function of fucosylation in cancer is to mediate metastasis through selectin ligand-dependent processes. Previously, we found complete loss of fucosylation in the colon cancer cell line HCT116 due to a mutation in the GDP-fucose synthetic enzyme, GDP-mannose-4,6-dehydratase (GMDS). Loss of fucosylation led to escape of cancer cells from tumor immune surveillance followed by tumor progression and metastasis, suggesting a novel function of fucosylation in tumor progression pathway. In the present study, we investigated the frequency of GMDS mutation in a number of clinical colorectal cancer tissue samples: 81 samples of primary colorectal cancer tissue and 39 samples of metastatic lesion including liver and lymph node. Four types of deletion mutation in GMDS were identified in original cancer tissues as well as metastatic lesions. The frequency of GMDS mutation was slightly higher in metastatic lesions (12.8%, 5/39 samples) than in original cancer tissues (8.6%, 7/81 samples). No mutation of the GMDS gene was observed in normal colon tissues surrounding cancer tissues, suggesting that the mutation is somatic rather than in the germline. Immunohistochemical analysis revealed complete loss of fucosylation in three cases of cancer tissue. All three cases had GMDS mutation. In one of three cases, loss of fucosylation was observed in only metastatic lesion, but not its original colon cancer tissue. These data demonstrate involvement of GMDS mutation in the progression of colorectal cancer.

Apoptosis and necrosis are two major forms of cell death observed in normal and disease pathologies. Although there are many assays for detection of apoptosis, relatively few assays are available for measuring necrosis. A key signature for necrotic cells is the permeabilization of the plasma membrane. This event can be quantified in tissue culture settings by measuring the release of the intracellular enzyme lactate dehydrogenase (LDH). When combined with other methods, measuring LDH release is a useful method for the detection of necrosis. In this chapter, we describe the step-by-step procedure for detection of LDH release from necrotic cells using a microtiter plate-based colorimetric absorbance assay. Fil: Chan, Francis Ka Ming. University of Massachusetts Medical School; Estados Unidos Fil: Moriwaki, Kenta. University of Massachusetts Medical School; Estados Unidos Fil: de Rosa, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. University of Massachusetts Medical School; Estados Unidos

Fucα1-6 oligosaccharide has a variety of biological functions and serves as a biomarker for hepatocellular carcinoma because of the elevated presence of fucosylated α-fetoprotein (AFP) in this type of cancer. In this study we purified a novel Fucα1-6-specific lectin from the mushroom Pholiota squarrosa by ion-exchange chromatography and affinity chromatography on thyroglobulin-agarose. The purified lectin was designated as PhoSL (P. squarrosa lectin). SDS-PAGE, MALDI-TOF mass spectrometry, and N-terminal amino acid sequencing indicate that PhoSL has a molecular mass of 4.5 kDa and consists of 40 amino acids (NH(2)-APVPVTKLVCDGDTYKCTAYLDFGDGRWVAQWDTNVFHTG-OH). Isoelectric focusing of the lectin showed bands near pI 4.0. The lectin activity was stable between pH 2.0 and 11.0 and at temperatures ranging from 0 to 100 °C for incubation times of 30 min. When PhoSL was investigated with frontal affinity chromatography using 132 pyridylaminated oligosaccharides, it was found that the lectin binds only to core α1-6-fucosylated N-glycans and not to other types of fucosylated oligosaccharides, such as α1-2-, α1-3-, and α1-4-fucosylated glycans. Furthermore, PhoSL bound to α1-6-fucosylated AFP but not to non-fucosylated AFP. In addition, PhoSL was able to demonstrate the differential expression of α1-6 fucosylation between primary and metastatic colon cancer tissues. Thus, PhoSL will be a promising tool for analyzing the biological functions of α1-6 fucosylation and evaluating Fucα1-6 oligosaccharides as cancer biomarkers.

Fucosylated alpha-fetoprotein (AFP) is a more specific biomarker for hepatocellular carcinoma (HCC) than AFP. However, the mechanisms underlying the increase in fucosylated AFP in sera of HCC patients remain largely unknown. Recently, we reported that fucosylation is a possible signal for the secretion of hepatic glycoproteins into bile and that the fucosylation-based sorting machinery might be disrupted in the liver bearing HCC. In this study, we investigated the selective secretion of fucosylated AFP into bile canaliculus (BC) structures of the human hepatoma cell line HepG2. The proportion of fucosylated AFP in BC structures was higher than that in the medium, as judged by lectin affinity electrophoresis. Suppression of fucosylation by the double knock-down of GDP-mannose-4,6-dehydratase and the human homologue of GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, which contribute to the synthesis of GDP-fucose, a donor substrate for fucosyltransferases, did not decrease the proportion of fucosylated AFP in BC structures but decreased this proportion in conditioned medium. Furthermore, increased AFP fucosylation was observed in medium, but not in BC structures, upon adding free fucose. These results suggest that saturation of fucosylated AFP in BC structures is accompanied by its increase in conditioned medium, probably leading to increased fucosylated AFP in sera of HCC patients.

Golgi phosphoprotein 2 (GP73) is a type II Golgi protein, which was found on examination of the fucosylated proteome as a potential tumor marker for hepatocellular carcinoma (HCC). The serum levels of both total and fucosylated GP73 were increased in the sera of patients with HCC. Fucosylation is one of the most important oligosaccharide modifications involved in cancer and is catalyzed by α1,6-fucosyltransferase (Fut8). In the present study, we investigated the effect of Fut8 overexpression on GP73 production in the human hepatoma cell line Hep3B. The Fut8 expression vector was transfected into Hep3B cells and the expression of GP73 was investigated by Western blotting and real-time PCR. Overexpression of Fut8 dramatically enhanced the expression of GP73 at the transcriptional level. Surprisingly, this effect was not dependent on cellular fucosylation. Overexpression of a mutant Fut8, which was unable to be localized to the Golgi, did not induce GP73 production, suggesting that the localization of Fut8 in the Golgi apparatus was important for the increase in GP73 expression. This is the first demonstration of GP73 regulation through overexpression of a glycosyltransferase, which may lead to Golgi stress.

Glycans exhibit characteristic changes in their structures during development and thus have been used as markers for stem/progenitor cells. However, the glycan structures unique to cancer stem cells (CSC) remain unknown. In the present study, we examined glycan structures in CD133+ CD13+ CSC, which were recently found to have a high CSC ability, by means of a lectin microarray. Seven sialylated glycan-recognizing lectins, MAL-I, SNA, SSA, TJA-I, ACG, ABA and MAH, showed higher affinity to CD133+ CD13+ CSC than CD133+ cells with a lower CSC ability. In addition, we demonstrated that CD133+ SSA+ cells isolated from Huh7 cells had a significantly higher ability to form tumors in non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice and spheres under serum-free conditions than CD133+ SSA- cells. These results suggest that hepatic CSC highly express sialylated glycans and that SSA lectin can be used as a tool for isolating CSC. This study is the first report to demonstrate the characteristic glycan structures in CSC and to indicate a new methodology involving lectins for isolating CSC.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many cancer cells but not in normal ones. Recombinant TRAIL and agonistic antibodies to its cognate receptors are currently being studied as promising anticancer drugs. However, preclinical and clinical studies have shown that many types of human cancers are resistant to TRAIL agonists. We previously reported that a deficiency of fucosylation, which is one of the most common oligosaccharide modifications, leads to resistance to TRAIL-induced apoptosis. In contrast, DNA methylation is associated with silencing of various tumor suppressor genes and resistance of cancer cells to anticancer drugs. The aim of this study is to clarify the involvement of DNA methylation in the regulation of cellular fucosylation and the susceptibility to TRAIL-induced apoptosis. When nineteen cancer cell lines with relatively low fucosylation levels were treated with a novel methyltransferase inhibitor, zebularine, an increase in the fucosylation level was observed in many cancer cell lines. The expression of fucosylation-related genes, such as the FX, GDP-fucose transporter, and Fut4 genes, was significantly increased after the treatment with zebularine. Moreover, a synergistic effect of zebularine on TRAIL-induced apoptosis was observed in several cancer cell lines, in which fucosylation was increased by treatment with zebularine. This synergistic effect was independent of the expression of TRAIL receptors and caspase-8. These results indicate that cellular fucosylation is regulated through DNA methylation in many cancer cells. Moreover, zebularine might be useful as a combination drug with TRAIL-based therapies in patients with TRAIL-resistant cancer.

Fucosylation is one of the most important oligosaccharide modifications in cancer and inflammation. The fucosylation level is increased in total cellular proteins of cancer cells as well as in sera of patients with cancer. Recently, on AAL blot analysis, we found a fucosylated glycoprotein of 40 kDa in sera of patients with pancreatic cancer. Based on its N-terminal sequence, this protein was identified as haptoglobin. Fucosylated haptoglobin was increased in sera of patients with several kinds of cancer and the positive rate was higher in pancreatic cancer. The level of fucosylated haptoglobin was not correlated with total haptoglobin, suggesting that a factor other than inflammation could regulate the production of fucosylated haptoglobin. Mass spectrometry analysis revealed the detailed oligosaccharide structure of fucosylated haptoglobin purified from sera of patients with pancreatic cancer. For clinical applications, we developed a lectin-antibody ELISA system for quantifying fucosylated haptoglobin. In this review, we would like to summarize the history of the identification of fucosylated haptoglobin as a marker for pancreatic cancer and its possible application for a clinical diagnostic test.

Oligosaccharides serve as markers of the cell surface and have been used as certain kinds of tumor markers. In the present study, we established a simple method for isolating hepatic progenitor cells using a lectin, which recognizes a characteristic oligosaccharide structure. Rat liver epithelial (RLE) cells, which have been established as a hepatic stem-like cell, were used to identify characteristic oligosaccharide structures on hepatic stem cells. As a result from lectin micro array, several types of lectin including E4-PHA were identified to bind RLE cells specifically. Furthermore, lectin blot and lectin flow cytometry analyses showed that binding to E(4)-PHA lectin was significantly increased in RLE cells, compared to hepatocytes, and hepatoma cells. The induction of differentiation into a hepatocyte lineage of RLE cells by treatment with Oncostatin M and dexamethasone resulted in a decrease in E(4)-PHA binding. Using an E(4)-PHA column, we succeeded in isolating hepatic stem cells from LEC (Long-Evans with cinnamon coat color) rat livers with fluminant hepatitis. The characteristics of the established cells were similar to RLE cells and had a potential of proliferating in rat liver. These results suggest that oligosaccharides can serve as a novel marker for the isolation of the hepatic progenitor cells.

Changes in the levels of fucosylation regulate the biological phenotype of cancer cells and a specific fucosylation, such as fucosylated alpha-fetoprotein (AFP-L3) has been clinically used as a tumor marker for hepatocellular carcinoma (HCC). However, detailed molecular mechanisms that explain the increased fucosylation in HCC remain unknown despite 10 years of study by these researchers. Fucosylation is regulated by complicated mechanisms that involve several factors: fucosyltransferases, GDP-fucose transporter (GDP-Fuc Tr), and synthetic enzymes of GDP-fucose, such as GDP-mannose 4, 6-dehydratase (GMD), GDP-4-keto-6-deoxy-mannose-3, 5-epimerase-4-reductase (FX), and GDP-fucose pyrophosphorylase. In this study, the expression of fucosylation-related genes in HCC tissues was studied and it was found that GDP-Fuc Tr is a key factor for increases in fucosylation. A real-time reverse transcription polymerase chain reaction (RT-PCR) analysis showed significant increases in GDP-Fuc Tr and FX expression in HCC, and levels of the GMD protein were upregulated by posttranslational modification in HCC tissues. In vitro cell experiments showed that the level of GDP-Fuc Tr was the most significantly correlated with the level of cellular fucosylation and the overexpression of GDP-Fuc Tr dramatically increased fucosylation in Hep3B cells. The importance of GDP-Fuc Tr in the increase of fucosylation was also confirmed with immunohistochemical analyses. These findings suggest that the upregulation of GDP-Fuc Tr plays a pivotal role in increased fucosylation in HCC and represents an attractive target for new treatments and diagnosis for HCC.

Apoptosis is a key mechanism for metazoans to eliminate unwanted cells. Resistance to apoptosis is a hallmark of many cancer cells and a major roadblock to traditional chemotherapy. Recent evidence indicates that inhibition of caspase-dependent apoptosis sensitizes many cancer cells to a form of non-apoptotic cell death termed necroptosis. This has led to widespread interest in exploring necroptosis as an alternative strategy for anti-cancer therapy. Here we show that in human colon cancer tissues, the expression of the essential necroptosis adaptors receptor interacting protein kinase (RIPK)1 and RIPK3 is significantly decreased compared with adjacent normal colon tissues. The expression of RIPK1 and RIPK3 was suppressed by hypoxia, but not by epigenetic DNA modification. To explore the role of necroptosis in chemotherapy-induced cell death, we used inhibitors of RIPK1 or RIPK3 kinase activity, and modulated their expression in colon cancer cell lines using short hairpin RNAs. We found that RIPK1 and RIPK3 were largely dispensable for classical chemotherapy-induced cell death. Caspase inhibitor and/or second mitochondria-derived activator of caspase mimetic, which sensitize cells to RIPK1- and RIPK3-dependent necroptosis downstream of tumor necrosis factor receptor-like death receptors, also did not alter the response of cancer cells to chemotherapeutic agents. In contrast to the RIPKs, we found that cathepsins are partially responsible for doxorubicin or etoposide-induced cell death. Taken together, these results indicate that traditional chemotherapeutic agents are not efficient inducers of necroptosis and that more potent pathway-specific drugs are required to fully harness the power of necroptosis in anti-cancer therapy.

Changes in oligosaccharide structures have been reported in certain types of malignant transformations and, thus, could be used for tumor markers in certain types of cancer. In the case of pancreatic cancer cell lines, a variety of fucosylated proteins are secreted into their conditioned media. To identify fucosylated proteins in the serum of patients with pancreatic cancer, we performed western blot analyses using Aleuria Aurantica Lectin (AAL), which is specific for fucosylated structures. An approximately 40 kD protein was found to be highly fucosylated in pancreatic cancer and an N-terminal analysis revealed that it was the beta chain of haptoglobin. While the appearance of fucosylated haptoglobin has been reported in other diseases such as hepatocellular carcinoma, liver cirrhosis, gastric cancer and colon cancer, the incidence was significantly higher in the case of pancreatic cancer. Fucosylated haptoglobin was observed more frequently at the advanced stage of pancreatic cancer and disappeared after an operation. A mass spectrometry analysis of haptoglobin purified from the serum of patients with pancreatic cancer and the medium from a pancreatic cancer cell line, PSN-1, showed that the alpha 1-3/alpha 1-4/alpha 1-6 fucosylation of haptoglobin was increased in pancreatic cancer. When a hepatoma cell line, Hep3B, was cultured with the conditioned media from pancreatic cancer cells, haptoglobin secretion was dramatically increased. These findings suggest that fucosylated haptoglobin could serve as a novel marker for pancreatic cancer. Two possibilities were considered in terms of the fucosylation of haptoglobin. One is that pancreatic cancer cells, themselves, produce fucosylated haptoglobin; the other is that pancreatic cancer produces a factor, which induces the production of fucosylated haptoglobin in the liver.

A growing body of evidence has suggested that tumors are frequently composed of heterogeneous cell types, and that tumor initiation and growth are driven by a small subset of cells, termed cancer stem cells (CSCs) or tumor-initiating cells. CSCs can self-renew and also give rise to more differentiated progeny that comprise the bulk of a tumor. Glycans exhibit characteristic changes in their structures during development and thus have been used as markers for stem/progenitor cells. However, the glycan structures unique to CSCs have remained unknown. In this study, we examined glycan structures in CD133+CD13+ CSCs, which were recently found to have a high CSC ability, by means of lectin microarray. Seven sialylated glycan-recognizing lectins, MAL-I, SNA, SSA, TJA-I, ACG, ABA, and MAH, showed higher affinity to CD133+CD13+ CSCs than CD133+ cells with a lower CSC ability. In addition, we demonstrated that CD133+SSA+ cells isolated from Huh7 cells had a significantly higher ability to form tumors in NOD/SCID mice and spheres under serum-free conditions than CD133+SSA- cells. These results suggest that hepatic CSCs highly express sialylated glycans and that SSA lectin can be used as a tool for isolating CSCs. This study is the first report to demonstrate the characteristic glycan structures in CSCs and to indicate a new methodology involving lectins for isolating CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4356. doi:10.1158/1538-7445.AM2011-4356

Oligosaccharides are some of the most important factors in the posttranslational modification of proteins and lipids. It is well known that oligosaccharide structures change during malignant transformation. Fucose is a type of constituent of oligosaccharides, and is notably associated with cancer and inflammation. Fucosylation is catalyzed by several kinds of fucosyltransferases, the GDP-fucose synthetic enzymes, and GDP-fucose transporter. Recently, we found a mutation of the GMDS gene, which involved in the synthesis of GDP-fucose, in a colon cancer cell line, HCT116, leading to a virtually complete deficiency of fucosylation. Down-regulation of cellular fucosylation rendered the cells resistant to tumor necrosis factor-related apotosis-inducing ligand (TRAIL) -induced apoptosis, resulting in the escape from natural killer (NK) cell-mediated tumor immune surveillance. Thus, GMDS and fucosylation are thought to be regulatory factors for TRAIL-induced apoptosis and tumor immune surveillance. In the present study, we searched mutation of the GMDS gene in human cancer cell lines and various kinds of cancer tissues. The aberrant transcripts with deletions of exons 2-4 and 5-7 were detected in the cDNA of LS174T and NCI-H716 cells, respectively. Since these mutations were heterozygous, the deficiency of fucosylation was not observed. In a gastric choriocarcinoma cell line, SCH, only short transcript with deletion of exons 2-4 was detected. This cell line showed a marked decrease in the fucosylation level. PCR and subsequent direct sequencing analyses using 100 human colon cancer tissues identified mutations in two cases. One of the two was c.739 C>T (p.R247X) in exon7, and the other was c.345+5 G>A (IVS4+5) in intron 4. Furthermore, RT-PCR analysis identified aberrant transcripts in 5 of 10 ovarian cancer tissues. All mutant GMDS genes found in this study had no enzymatic activity. Subsequently, the level of fucosylation in many colon cancer tissues was investigated by immunohistochemical analysis using two types of fucosylated oligosaccharide-specific lectin. The decrease of fucosylation was observed in certain cases. Especially, core-fucosylation was decreased in colon cancer with lymph node metastasis. These results suggest that cancer progression in certain patients with colon cancer is dependent on de-fucosylation. Thus, fucosylation is an attractive target for cancer diagnosis and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 800.

Background & Aims Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) promotes apoptosis in cancer cells, but not normal cells, and is critically involved in tumor rejection through natural killer (NK) cell–mediated immune surveillance. Oligosaccharides are involved in various aspects in carcinogenesis, and fucosylation is one of the most important oligosaccharide modifications in cancer. Here, we report for the first time mutations of the GDP-mannose-4,6-dehydratase ( GMDS ) gene, which plays a pivotal role in fucosylation, in human colon cancer. The mutations resulted in resistance to TRAIL-induced apoptosis followed by escape from immune surveillance. Methods The mock and GMDS -rescued HCT116 cells were investigated in terms of NK cell–mediated tumor surveillance by TRAIL signaling both in vitro and in vivo. The mutational analysis for GMDS was performed with kinds of cancer cell lines and tissues. Results The mutation found here led to a virtually complete deficiency of cellular fucosylation, and transfection of the wild-type GMDS into HCT116 cells restored the cellular fucosylation. When mock and GMDS -rescued cells were transplanted into athymic mice, tumor growth and metastasis of the GMDS -rescued cells were dramatically suppressed through NK cell–mediated tumor surveillance. Furthermore, the GMDS -rescued cells showed high susceptibility to TRAIL-induced apoptosis, and anti-TRAIL blocking antibody suppressed the accelerated direct cell lysis of the GMDS -rescued cells by splenocytes. Similar mutations of the GMDS were found in certain human cancer tissues and other cell lines. Conclusions This pathway by GMDS mutation could be a novel type of cancer progression through cellular fucosylation and NK cell–mediated tumor surveillance.