Fucosylation inhibitor 6-alkynylfucose enhances the ATRA-induced differentiation effect on acute promyelocytic leukemia cells.

For acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) is well established. However, the narrow application and tolerance development of ATRA remain to be improved. In this study, we investigated the effects of combinations of glycosylation inhibitors with ATRA to achieve better efficiency than ATRA alone. We found that the combination of fucosylation inhibitor 6-alkynylfucose (6AF) and ATRA had an additional effect on cell differentiation, as revealed by expression changes in two differentiation markers, CD11b and CD11c, and significant morphological changes in NB4 APL and HL-60 acute myeloid leukemia (AML) cells. In AAL lectin blot analyses, ATRA or 6AF alone could decrease fucosylation, while their combination decreased fucosylation more efficiently. To clarify the molecular mechanism for the 6AF effect on ATRA-induced differentiation, we performed microarray analyses using NB4 cells. In a pathway analysis using DAVID software, we found that the C-type lectin receptor (CLR) signaling pathway was enriched with high significance. In real-time PCR analyses using NB4 and HL-60 cells, FcεRIγ, CLEC6A, CLEC7A, CASP1, IL-1ß, and EGR3, as components of the CLR pathway, as well as CD45 and AKT3 were upregulated by 6AF in ATRA-induced differentiation. Taken together, the present findings suggest that the CLR signaling pathway is involved in the 6AF effect on ATRA-induced differentiation.

BRCC36 associates with FLT3-ITD to regulate its protein stability and intracellular signaling in acute myeloid leukemia.

Fms-like tyrosine kinase-3 (FLT3) is a commonly mutated gene in acute myeloid leukemia (AML). The two most common mutations are the internal-tandem duplication domain (ITD) mutation and the tyrosine kinase domain (TKD) mutation. FLT3-ITD and FLT3-TKD exhibit distinct protein stability, cellular localization, and intracellular signaling. To understand the underlying mechanisms, we performed proximity labeling with TurboID to identify proteins that regulate FLT3-ITD or -TKD differently. We found that BRCA1/BRCA2-containing complex subunit 36 (BRCC36), a specific K63-linked polyubiquitin deubiquitinase, was exclusively associated with ITD, not the wild type of FLT3 and TKD. Knockdown of BRCC36 resulted in decreased signal transducers and activators of transcription 5 phosphorylation and cell proliferation in ITD cells. Consistently, treatment with thiolutin, an inhibitor of BRCC36, specifically suppressed cell proliferation and induced cell apoptosis in ITD cells. Thiolutin efficiently affected leukemia cell lines expressing FLT3-ITD cell viability and exhibited mutual synergies with quizartinib, a standard clinical medicine for AML. Furthermore, mutation of the lysine at 609 of ITD led to significant suppression of K63 polyubiquitination and decreased its stability, suggesting that K609 is a critical site for K63 ubiquitination specifically recognized by BRCC36. These data indicate that BRCC36 is a specific regulator for FLT3-ITD, which may shed light on developing a novel therapeutic approach for AML.

Exogenous l-fucose attenuates neuroinflammation induced by lipopolysaccharide.

α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of fucose to the innermost GlcNAc residue of N-glycan to form core fucosylation. Our previous studies showed that lipopolysaccharide (LPS) treatment highly induced neuroinflammation in Fut8 homozygous KO (Fut8-/-) or heterozygous KO (Fut8+/-) mice, compared with the WT (Fut8+/+) mice. To understand the underlying mechanism, we utilized a sensitive inflammation-monitoring mouse system that contains the human interleukin-6 (hIL6) bacterial artificial chromosome transgene modified with luciferase (Luc) reporter cassette. We successfully detected LPS-induced neuroinflammation in the central nervous system by exploiting this bacterial artificial chromosome transgenic monitoring system. Then we examined the effects of l-fucose on neuroinflammation in the Fut8+/- mice. The lectin blot and mass spectrometry analysis showed that l-fucose preadministration increased the core fucosylation levels in the Fut8+/- mice. Notably, exogenous l-fucose attenuated the LPS-induced IL-6 mRNA and Luc mRNA expression in the cerebral tissues, confirmed using the hIL6-Luc bioluminescence imaging system. The activation of microglial cells, which provoke neuroinflammatory responses upon LPS stimulation, was inhibited by l-fucose preadministration. l-Fucose also suppressed the downstream intracellular signaling of IL-6, such as the phosphorylation levels of JAK2 (Janus kinase 2), Akt (protein kinase B), and STAT3 (signal transducer and activator of transcription 3). l-Fucose administration increased gp130 core fucosylation levels and decreased the association of gp130 with the IL-6 receptor in Fut8+/- mice, which was further confirmed in BV-2 cells. These results indicate that l-fucose administration ameliorates the LPS-induced neuroinflammation in the Fut8+/- mice, suggesting that core fucosylation plays a vital role in anti-inflammation and that l-fucose is a potential prophylactic compound against neuroinflammation.

A highly specific antibody against the core fucose of the N-glycan in IgG identifies the pulmonary diseases and its regulation by CCL2.

Glycan structure is often modulated in disease or predisease states, suggesting that such changes might serve as biomarkers. Here, we generated a monoclonal antibody (mAb) against the core fucose of the N-glycan in human IgG. Notably, this mAb can be used in Western blotting and ELISA. ELISA using this mAb revealed a low level of the core fucose of the N-glycan in IgG, suggesting that the level of acore fucosylated (noncore fucosylated) IgG was increased in the sera of the patients with lung cancer, chronic obstructive pulmonary disease, and interstitial pneumonia compared to healthy subjects. In a coculture analysis using human lung adenocarcinoma A549 cells and antibody-secreting B cells, the downregulation of the FUT8 (α1,6 fucosyltransferase) gene and a low level of core fucose of the N-glycan in IgG in antibody-secreting B cells were observed after coculture. A dramatic alteration in gene expression profiles for cytokines, chemokines, and their receptors were also observed after coculturing, and we found that the identified C-C motif chemokine 2 was partially involved in the downregulation of the FUT8 gene and the low level of core fucose of the N-glycan in IgG in antibody-secreting B cells. We also developed a latex turbidimetric immunoassay using this mAb. These results suggest that communication with C-C motif chemokine 2 between lung cells and antibody-secreting B cells downregulate the level of core fucose of the N-glycan in IgG, i.e., the increased level of acore fucosylated (noncore fucosylated) IgG, which would be a novel biomarker for the diagnosis of patients with pulmonary diseases.

Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway.

The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway.

Bone morphogenetic protein signaling is a possible therapeutic target in gynecologic cancer.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor ß (TGFß) superfamily. BMPs play crucial roles in embryogenesis and bone remodeling. Recently, BMP signaling has been found to have diverse effects on different types of tumors. In this review, we summarized the effects of BMP signaling on gynecologic cancer. BMP signaling has tumor-promoting effects on ovarian cancer (OC) and endometrial cancer (EC), whereas it has tumor-suppressing effects on uterine cervical cancer (UCC). Interestingly, EC has frequent gain-of-function mutations in ACVR1, encoding one of the type I BMP receptors, which are also observed in fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma. Little is known about the relationship between BMP signaling and other gynecologic cancers. Tumor-promoting effects of BMP signaling in OC and EC are dependent on the promotion of cancer stemness and epithelial-mesenchymal transition (EMT). In accordance, BMP receptor kinase inhibitors suppress the cell growth and migration of OC and EC. Since both cancer stemness and EMT are associated with chemoresistance, BMP signaling activation might also be an important mechanism by which OC and EC patients acquire chemoresistance. Therefore, BMP inhibitors are promising for OC and EC patients even if they become resistant to standard chemotherapy. In contrast, BMP signaling inhibits UCC growth in vitro. However, the in vivo effects of BMP signaling have not been elucidated in UCC. In conclusion, BMP signaling has a variety of functions, depending on the types of gynecologic cancer. Therefore, targeting BMP signaling should improve the treatment of patients with gynecologic cancer.

The Histone Methyltransferase SETD8 Regulates the Expression of Tumor Suppressor Genes via H4K20 Methylation and the p53 Signaling Pathway in Endometrial Cancer Cells.

The histone methyltransferase SET domain-containing protein 8 (SETD8), which methylates histone H4 lysine 20 (H4K20) and non-histone proteins such as p53, plays key roles in human carcinogenesis. Our aim was to determine the involvement of SETD8 in endometrial cancer and its therapeutic potential and identify the downstream genes regulated by SETD8 via H4K20 methylation and the p53 signaling pathway. We examined the expression profile of SETD8 and evaluated whether SETD8 plays a critical role in the proliferation of endometrial cancer cells using small interfering RNAs (siRNAs). We identified the prognostically important genes regulated by SETD8 via H4K20 methylation and p53 signaling using chromatin immunoprecipitation sequencing, RNA sequencing, and machine learning. We confirmed that SETD8 expression was elevated in endometrial cancer tissues. Our in vitro results suggest that the suppression of SETD8 using siRNA or a selective inhibitor attenuated cell proliferation and promoted the apoptosis of endometrial cancer cells. In these cells, SETD8 regulates genes via H4K20 methylation and the p53 signaling pathway. We also identified the prognostically important genes related to apoptosis, such as those encoding KIAA1324 and TP73, in endometrial cancer. SETD8 is an important gene for carcinogenesis and progression of endometrial cancer via H4K20 methylation.

Multifaceted Functions of TWSG1: From Embryogenesis to Cancer Development.

Bone morphogenetic proteins (BMPs) play an important role in development. Twisted gastrulation BMP signaling modulator 1 (TWSG1) was initially identified as a regulator of the dorsoventral axis formation in Drosophila. The mechanism of BMP signaling modulation by TWSG1 is complex. TWSG1 inhibits BMP signaling by binding to BMP ligands including BMP4, whereas it enhances signaling by interacting with Chordin, a BMP antagonist. Therefore, TWSG1 can act as both a BMP agonist and antagonist. TWSG1 has various functions ranging from embryogenesis to cancer progression. TWSG1 knockout mice showed neural, craniofacial, and mammary defects. TWSG1 also regulated erythropoiesis and thymocyte development. Furthermore, the relationship between TWSG1 and cancer has been elucidated. Allelic loss of TWSG1 was detected in colorectal cancer. TWSG1 expression was upregulated in papillary thyroid carcinoma and glioblastoma but downregulated in gastric and endometrial cancers. TWSG1 suppressed BMP7-enhanced sphere formation and migration in endometrial cancer cells, indicating its tumor-suppressive role. Further studies are required to clarify the TWSG1 function and its association with BMP signaling in cancer development. Finally, TWSG1 is abundantly expressed in human and mouse ovaries and sustains follicular growth in rodent ovaries. Thus, TWSG1 has various functions ranging from fertility to cancer. Therefore, TWSG1 signaling modulation may be beneficial in treating specific diseases such as cancer.

The Two-Faced Role of Autophagy in Endometrial Cancer.

Autophagy, meaning "self-eating," is a cellular catabolic process that involves lysosomal degradation of cytoplasmic materials. Autophagy contributes to both quality control and energy supply of cells, which are associated with tumorigenesis and tumor development, respectively. Endometrial cancer (EC) is the most common gynecologic cancer, and its incidence is increasing. Although autophagy plays crucial roles in several types of cancer, such as pancreatic ductal adenocarcinoma, its role in EC has not been clearly demonstrated. Activation of the PI3K/AKT/mTOR pathway, which functions to suppress autophagy, is an initial step in type 1 endometrial carcinogenesis, whereas a loss-of-function mutation of TP53, which augments autophagy via p16 induction, is the main cause of type 2 endometrial carcinogenesis. Mutations in autophagy-related genes, including ATG4C, RB1CC1/FIP200, and ULK4, have been reported in EC; thus, an aberrant autophagy mechanism may be involved in endometrial carcinogenesis. Furthermore, the biguanide diabetes drug metformin, treatment with which enhances autophagy via AMPK-mediated mTOR inactivation, has been reported to reduce the risk of EC. These findings suggest that autophagy negatively regulates endometrial carcinogenesis, and autophagy inducers may be useful for chemoprevention of EC. In contrast, autophagy appears to promote EC once it is established. Consistent with this, treatment with chloroquine, an autophagy inhibitor, is reported to attenuate EC cell proliferation. Moreover, chemotherapy-induced autophagy triggers chemoresistance in EC cells. As autophagy has a tumor-promoting function, the combination of chemotherapy and autophagy inhibitors such as chloroquine could be a potent therapeutic option for patients with EC. In conclusion, autophagy plays a dual role in the prevention and treatment of EC. Therefore, targeting autophagy to prevent and treat EC requires diametrically opposed strategies.

α-1,6-Fucosyltransferase Is Essential for Myogenesis in Zebrafish.

Glycosylation is an important mechanism regulating various biological processes, including intercellular signaling and adhesion. α-1,6-fucosyltransferase (Fut8) belongs to a family of enzymes that determine the terminal structure of glycans. Fut8 is widely conserved from Caenorhabditis elegans to humans, and its mutants have been reported in humans, mice, and zebrafish. Although mutants show various symptoms, such as spinal deformity and growth retardation, its effects on skeletal muscles are unknown. We aimed to elucidate the function of Fut8 in skeletal muscle using zebrafish and C2C12 cells for evaluation. We observed that most fut8a morphants died at 2 days post-fertilization (dpf) or in earlier developmental stages even at low concentrations of morpholino oligonucleotides (MOs). Mutant juveniles also had small body sizes, and abnormal myocepta and sarcomere structures, suggesting that Fut8a plays important roles in myogenesis. Moreover, treatment of C2C12 cells with 2-fluorofucose (2FF), a fucosylation inhibitor, during cell differentiation dramatically reduced the expression of myogenic genes, such as Myomaker and other myogenic fusion genes, and inhibited myotube formation. These results indicate that Fut8 is an important factor in myogenesis, and myofusion in particular.

BMP2-induction of FN14 promotes protumorigenic signaling in gynecologic cancer cells.

We previously reported that bone morphogenetic protein (BMP) signaling promotes tumorigenesis in gynecologic cancer cells. BMP2 enhances proliferation of ovarian and endometrial cancer cells via c-KIT induction, and triggers epithelial-mesenchymal transition (EMT) by SNAIL and/or SLUG induction, leading to increased cell migration. However, the downstream effectors of BMP signaling in gynecological cancer cells have not been clearly elucidated. In this study, we performed RNA-sequencing of Ishikawa endometrial and SKOV3 ovarian cancer cells after BMP2 stimulation, and identified TNFRSF12A, encoding fibroblast growth factor-inducible 14 (FN14) as a common BMP2-induced gene. FN14 knockdown suppressed BMP2-induced cell proliferation and migration, confirmed by MTS and scratch assays, respectively. In addition, FN14 silencing augmented chemosensitivity of SKOV3 cells. As a downstream effector of BMP signaling, FN14 modulated both c-KIT and SNAIL expression, which are important for growth and migration of ovarian and endometrial cancer cells. These results support the notion that the tumor promoting effects of BMP signaling in gynecological cancers are partially attributed to FN14 induction.

Tumor Promoting Effect of BMP Signaling in Endometrial Cancer.

The effects of bone morphogenetic proteins (BMPs), members of the transforming growth factor-ß (TGF-ß) family, in endometrial cancer (EC) have yet to be determined. In this study, we analyzed the TCGA and MSK-IMPACT datasets and investigated the effects of BMP2 and of TWSG1, a BMP antagonist, on Ishikawa EC cells. Frequent ACVR1 mutations and high mRNA expressions of BMP ligands and receptors were observed in EC patients of the TCGA and MSK-IMPACT datasets. Ishikawa cells secreted higher amounts of BMP2 compared with ovarian cancer cell lines. Exogenous BMP2 stimulation enhanced EC cell sphere formation via c-KIT induction. BMP2 also induced EMT of EC cells, and promoted migration by induction of SLUG. The BMP receptor kinase inhibitor LDN193189 augmented the growth inhibitory effects of carboplatin. Analyses of mRNAs of several BMP antagonists revealed that TWSG1 mRNA was abundantly expressed in Ishikawa cells. TWSG1 suppressed BMP7-induced, but not BMP2-induced, EC cell sphere formation and migration. Our results suggest that BMP signaling promotes EC tumorigenesis, and that TWSG1 antagonizes BMP7 in EC. BMP signaling inhibitors, in combination with chemotherapy, might be useful in the treatment of EC patients.

α1,6-Fucosyltransferase contributes to cell migration and proliferation as well as to cancer stemness features in pancreatic carcinoma.

BACKGROUND: Pancreatic carcinoma is one of the deadliest malignant diseases, in which the increased expression of α1,6-fucosyltransferase (FUT8), a sole enzyme responsible for catalyzing core fucosylation, has been reported. However, its pathological roles and regulatory mechanisms remain largely unknown. Here, we use two pancreatic adenocarcinoma cell lines, MIA PaCa-2 and PANC-1 cells, as cell models, to explore the relationship of FUT8 with the malignant transformation of PDAC. METHODS: FUT8 knockout (FUT8-KO) cells were established by the CRISPR/Cas9 system. Cell migration was analyzed by transwell and wound-healing assays. Cell proliferation was examined by MTT and colony-formation assays. Cancer stemness markers and spheroid formations were used to analyzed cancer stemness features. RESULTS: Deficiency of FUT8 inhibited cell migration and proliferation in both MIA PaCa-2 and PANC-1 cells compared with wild-type cells. Moreover, the expression levels of cancer stemness markers such as EpCAM, CXCR4, c-Met, and CD133 were decreased in the FUT8-KO cells compared with wild-type cells. Also, the spheroid formations in the KO cells were loose and unstable, which could be reversed by restoration with FUT8 gene in the KO cells. Additionally, FUT8-KO increased the chemosensitivity to gemcitabine, which is the first-line therapy for advanced pancreatic cancer. CONCLUSIONS: FUT8-KO reduced the cell proliferation and migration. Our results are the first to suggest that the expression of FUT8 is involved in regulating the stemness features of pancreatic cancer cells. GENERAL SIGNIFICANCE: FUT8 could provide novel insights for the treatment of pancreatic carcinoma.

BMP signaling is a therapeutic target in ovarian cancer.

BMP signaling has been found to have tumor-promoting as well as tumor-suppressing effects in different types of tumors. In this study, we investigated the effects of BMP signaling and of BMP inhibitors on ovarian cancer (OC) cells in vitro and in vivo. High expression of BMP receptor 2 (BMPR2) correlated with poor overall survival of OC patients in the TCGA dataset. Both BMP2 and BMPR2 enhanced OC cell proliferation, whereas BMP receptor kinase inhibitors inhibited OC cell growth in cell culture as well as in a mouse model. BMP2 also augmented sphere formation, migration, and invasion of OC cells, and induced EMT. High BMP2 expression was observed after chemotherapy of OC patients in the GSE109934 dataset. In accordance, carboplatin, used for the treatment of OC patients, increased BMP2 secretion from OC cells, and induced EMT partially via activation of BMP signaling. Our data suggest that BMP signaling has tumor-promoting effects in OC, and that BMP inhibitors might be useful therapeutic agents for OC patients. Considering that carboplatin treatment augmented BMP2 secretion, the possibility to use a combination of BMP inhibitors and carboplatin in the treatment of OC patients, would be worth exploring.

Potato tuber metabolomics-based prediction of chip color quality and application using gas chromatography/flame ionization detector.

Potato (Solanum tuberosum L.) tubers are usually harvested once a year; thus, long-term storage is required to supply quality-assured tubers throughout the year. Further, an applicable method to predict tuber quality during storage is needed. In this study, gas chromatography-mass spectrometry (GC/MS) metabolomics was applied to identify applicable biomarkers for prediction of potato chip color based on 3 years' field-grown tubers. The projections to latent structures (PLS) prediction model, calculated from a metabolome data set obtained before storage, was consistent with actual measured chip color values. Additionally, GC with frame ionization detector (GC/FID) metabolite fingerprinting simultaneously re-constructed more reliable and relevant prediction models for chip color quality compared to GC/MS. Moreover, nine metabolites detected by GC/MS analysis were further validated as applicable prediction markers. This strategy will provide a practical and cost-effective quality-control tool for potato processing manufacturers on an industrial scale.

Loss of core fucosylation enhances the anticancer activity of cytotoxic T lymphocytes by increasing PD-1 degradation.

As an immune checkpoint, programmed cell death 1 (PD-1) and its ligand (PD-L1) pathway plays a crucial role in CD8+ cytotoxic T lymphocytes (CTL) activation and provides antitumor responses. The N-glycans of PD-1 and PD-L1 are highly core fucosylated, which are solely catalyzed by the core fucosyltransferase (Fut8). However, the precise biological mechanisms underlying effects of core fucosylation of PD-1 and PD-L1 on CTL activation have not been fully understood. In this study, we found that core fucosylation was significantly upregulated in lung adenocarcinoma. Compared to those of Fut8+/+ OT-I mice, the lung adenocarcinoma formation induced by urethane was markedly reduced in Fut8-/- OT-I mice. De-core fucosylation of PD-1 compromised its expression on Fut8-/- CTL, resulted in enhanced Fut8-/- CTL activation and cytotoxicity, leading to more efficient tumor eradication. Indeed, loss of core fucosylation significantly enhanced the PD-1 ubiquitination and in turn led to the degradation of PD-1 in the proteasome. Our current work indicates that inhibition of core fucosylation is a unique strategy to reduce PD-1 expression for the antilung adenocarcinoma immune therapy in the future.

Deficiency of core fucosylation activates cellular signaling dependent on FLT3 expression in a Ba/F3 cell system.

Fms-like tyrosine kinase 3 (FLT3) is a glycoprotein, that is a member of the class III receptor tyrosine kinase family. Approximately one-third of acute myeloid leukemia (AML) patients have mutations of this gene, and activation of the FLT3 downstream pathway plays an important role in both normal and malignant hematopoiesis. However, the role of N-glycosylation for FLT3 activation remains unclear. In this study, we showed that the N-glycan structures on wild type (WT), internal tandem duplication (ITD), and tyrosine kinase domain (TKD) mutants of FLT3 were different. Interestingly, expression of either WT or mutant FLT3 in Ba/F3 cells, an interleukin-3 (IL-3)-dependent hematopoietic progenitor cell, greatly induced core fucosylation. To elucidate the function of core fucosylation in FLT3-mediated signaling, we used a CRISPR/Cas9 system to establish α1,6-fucosyltransferase (Fut8) knockout (KO) cells. Surprisingly, the Fut8KO resulted in cell proliferation in an IL-3-independent manner in FLT3-WT cells, which was not observed in the parental cells, and suggested that this proliferation is dependent on FLT3 expression. Fut8KO greatly increased cellular tyrosine phosphorylation levels, together with an activation of STAT5, AKT, and ERK signaling, which could be completely neutralized by restoration with Fut8 in the KO cells. Consistently, a tyrosine kinase inhibitor efficiently inhibited cell proliferation induced by Fut8KO or specific fucosylation inhibitor. Additionally, immunostaining with FLT3 showed that the proteins were mainly expressed on the cell surface in the KO cells, which is similar to FLT3-WT cells, but different from the ITD mutant. Finally, we found that Fut8KO could induce dimer-formation in FLT3 without ligand-stimulation. Taken together, the present study clearly defines the regulatory function of core fucosylation in FLT3, which could provide a valuable direction for development of drugs could be effective in the treatment of AML.

EpCAM associates with integrin and regulates cell adhesion in cancer cells.

The epithelial cell adhesion molecule (EpCAM) is one of the most frequently and intensely expressed of tumor-associated antigens, but the role that EpCAM plays in the proliferation, adhesion and migration properties of cancer cells remains unclear. In the present study, we screened several tumor cell lines and found that colorectal cancer CW-2 and epidermoid carcinoma A431 cells expressed relatively higher levels of EpCAM. In order to assess the biological functions of EpCAM expression in cell adhesion and migration, we established a knock out (KO) of EpCAM genes in both of these types of cancer cells via a CRISPR/Cas9 system. The elongated cell morphology was converted to a rounded morphology in the EpCAM-KO cells. These cells showed decreases in cell proliferation and migration into extracellular matrix proteins, as well as decreases in cellular signaling elements such as phosphorylated focal adhesion kinase (FAK), AKT and ERK. Moreover, the cell growth and the colony formation abilities were significantly decreased in EpCAM-KO cells. Importantly, co-immunoprecipitation analysis revealed that EpCAM associated with integrin ß1. Also, the expression levels of integrin α5 were decreased in EpCAM-KO cells, compared with that in the wild-type cells. Taken together, these data clearly demonstrate that EpCAM associates with integrin ß1 to regulate FAK/ERK signaling pathways in controlling cell adhesion, migration and proliferation via extracellular matrix adhesion, which provides novel mechanisms for EpCAM-mediated biological functions and cancer phenotypes.

Identification of the minimal N-glycosylation on integrin α5ß1 required for its inhibitory effect on EGFR signaling and cell proliferation.

The N-glycosylation of integrin α5ß1 is involved in multiple cell biological functions. Our group previously reported that the N-glycosylation of the Calf-1,2 domain on α5 subunit (S3-5,10-14) was important for its inhibitory effect on EGFR signaling through regulating α5-EGFR complex formation. In this follow-up study, we provide evidence that the N-glycosylation on integrin ß1 subunit suppress cell growth by promoting its association with EGFR under fibronectin (FN)-coated conditions. Expression of wild-type (WT) ß1, but not the N-glycosylation mutant S4-6 ß1, which contains fewer N-glycans, inhibited EGFR signaling and cell proliferation after cell adhesion to FN. Furthermore, consistent restoration of the N-glycans on sites 1-3 of ß1 reinstated the inhibitory effects. Mechanistically, the N-glycosylation mutant of ß1 (S4-6+1-3) inhibited the EGFR response upon EGF stimulation via facilitating the α5ß1-EGFR complex formation. Moreover, we identified the N-glycosylation of sites 10-14 on α5 and 1-3 on ß1 were most important for EGFR signaling. Taken together, these data indicate that α5S3-5+10-14ß1S4-6+1-3 mutant represents the minimal N-glycosylation required for its regulation on EGFR signaling and cell proliferation, providing a plausible mechanism for the crosstalk between with α5ß1 and EGFR.

Gas chromatography-mass spectrometry metabolomics-based prediction of potato tuber sprouting during long-term storage.

In order to supply potato (Solanum tuberosum L.) tubers for the processed food industry throughout the year, suppliers should provide consistent quality potatoes even after long-term storage. Despite being one of the most important foods, there is no simple way to control tuber quality and, in particular, controlling sprouting. Chemical suppression such as chlorpropham is used to inhibit sprouting, however, the regulatory status of such chemical inhibition differs in each country. Gas chromatography-mass spectrometry-based metabolomics was applied to identify the applicable biomarkers for prediction of potato sprouting during long-term storage. Sprouting was measured in chipping potatoes, and these were also subjected to metabolite profiling to develop a predictive model. The model was based on projections to latent structures (PLS) regression calculated from a metabolome data set obtained before storage and was consistent with actual measured sprouting values. Sucrose, phosphate, and amino acids were selected as valid contributing biomarkers for prediction in a validation field experiment. These biomarkers will contribute to the development of a successful novel method for prediction and control of potato tuber quality during long-term storage.