Chiung-Hui Liu

Mucin glycosylating enzyme GALNT2 regulates the malignant character of hepatocellular carcinoma by modifying the EGF receptor.

Extracellular glycosylation is a critical determinant of malignant character. Here, we report that N-acetylgalactosaminyltransferase 2 (GALNT2), the enzyme that mediates the initial step of mucin type-O glycosylation, is a critical mediator of malignant character in hepatocellular carcinoma (HCC) that acts by modifying the activity of the epidermal growth factor receptor (EGFR). GALNT2 mRNA and protein were downregulated frequently in HCC tumors where these events were associated with vascular invasion and recurrence. Restoring GALNT2 expression in HCC cells suppressed EGF-induced cell growth, migration, and invasion in vitro and in vivo. Mechanistic investigations revealed that the status of the O-glycans attached to the EGFR was altered by GALNT2, changing EGFR responses after EGF binding. Inhibiting EGFR activity with erlotinib decreased the malignant characters caused by siRNA-mediated knockdown of GALNT2 in HCC cells, establishing the critical role of EGFR in mediating the effects of GALNT2 expression. Taken together, our results suggest that GALNT2 dysregulation contributes to the malignant behavior of HCC cells, and they provide novel insights into the significance of O-glycosylation in EGFR activity and HCC pathogenesis.

C1GALT1 Enhances Proliferation of Hepatocellular Carcinoma Cells via Modulating MET Glycosylation and Dimerization

Altered glycosylation is a hallmark of cancer. The core 1 β1,3-galactosyltransferase (C1GALT1) controls the formation of mucin-type O-glycans, far overlooked and underestimated in cancer. Here, we report that C1GALT1 mRNA and protein are frequently overexpressed in hepatocellular carcinoma tumors compared with nontumor liver tissues, where it correlates with advanced tumor stage, metastasis, and poor survival. Enforced expression of C1GALT1 was sufficient to enhance cell proliferation, whereas RNA interference-mediated silencing of C1GALT1 was sufficient to suppress cell proliferation in vitro and in vivo. Notably, C1GALT1 attenuation also suppressed hepatocyte growth factor (HGF)-mediated phosphorylation of the MET kinase in hepatocellular carcinoma cells, whereas enforced expression of C1GALT1 enhanced MET phosphorylation. MET blockade with PHA665752 inhibited C1GALT1-enhanced cell viability. In support of these results, we found that the expression level of phospho-MET and C1GALT1 were associated in primary hepatocellular carcinoma tissues. Mechanistic investigations showed that MET was decorated with O-glycans, as revealed by binding to Vicia villosa agglutinin and peanut agglutinin. Moreover, C1GALT1 modified the O-glycosylation of MET, enhancing its HGF-induced dimerization and activation. Together, our results indicate that C1GALT1 overexpression in hepatocellular carcinoma activates HGF signaling via modulation of MET O-glycosylation and dimerization, providing new insights into how O-glycosylation drives hepatocellular carcinoma pathogenesis.

MUC1 Expression Is Increased During Human Placental Development and Suppresses Trophoblast-Like Cell Invasion In Vitro

Abstract Mucin (MUC)1 is a multifunctional mucin expressed by a variety of reproductive tract epithelia. Trophoblast invasion is essential for normal placental development. However, MUC1 expression in the human placenta throughout pregnancy and the role of MUC1 in trophoblast-like cell invasion are still unclear. In the present study, results from quantitative RT-PCR and Western blot demonstrated that MUC1 mRNA and MUC1 protein expression, respectively, increased with gestational age of the human placenta. Immunohistochemistry revealed that MUC1 in placental villi was mainly expressed by syncytiotrophoblasts throughout pregnancy and increased with gestational age. Interestingly, we found two populations of extravillous trophoblasts, MUC1-positive and MUC1-negative cells, in decidua. The numbers of MUC1-positive extravillous trophoblasts were increased during placental development. Furthermore, MUC1 overexpression significantly (P < 0.01) suppressed matrigel invasion of trophoblast-like JAR cells by 34.6% ± 4.5% compared with control, which was associated with a decrease in MMP9 activity assessed by gelatin zymography. Our results suggest that MUC1 expression in the human placenta is increased during placental development, and its overexpression suppresses trophoblast-like cell invasion in vitro.

GALNT2 enhances migration and invasion of oral squamous cell carcinoma by regulating EGFR glycosylation and activity

OBJECTIVES Oral squamous cell carcinoma (OSCC) is one of the leading cancers worldwide. Aberrant glycosylation affects many cellular properties in cancers, including OSCC. This study aimed to explore the role of N-acetylgalactosaminyltransferase 2 (GALNT2) in OSCC. MATERIALS AND METHODS Immunohistochemistry was performed to study the expression of GALNT2 in an OSCC tissue microarray. Effects of GALNT2 overexpression and knockdown on cell migration and invasion were analyzed in SAS cells by transwell migration assay and matrigel invasion assay, respectively. The Vicia villosa agglutinin (VVA) pull down assay was conducted to detect changes in O-glycans on acceptor substrates of GALNT2. Cell signaling was analyzed by Western blotting. RESULTS GALNT2 was overexpressed in 73% (35/48) of OSCC tissues. Moreover, GALNT2 expression was localized in the invasive front and increased in high grade OSCC. GALNT2 overexpression enhanced migration and invasion of SAS cells triggered by fetal bovine serum (FBS) and epidermal growth factor (EGF). In contrast, GALNT2 knockdown inhibited SAS cell migration and invasion. Furthermore, GALNT2 overexpression enhanced VVA binding to epidermal growth factor receptor (EGFR) and EGF-induced phosphorylation of EGFR and AKT. Conversely, GALNT2 knockdown decreased VVA binding and suppressed activity of EGFR and AKT. CONCLUSION GALNT2 is frequently overexpressed in OSCC, especially in the carcinoma cells at the invasive front. GALNT2 overexpression enhances the invasive potential of OSCC cells via modifying O-glycosylation and activity of EGFR. These findings suggest that GALNT2 plays an important role in the invasive behavior of OSCC and that targeting GALNT2 could be a promising approach for OSCC therapy.

Neuregulin Facilitates Nerve Regeneration by Speeding Schwann Cell Migration via ErbB2/3-Dependent FAK Pathway

Background Adequate migration of Schwann cells (Sc) is crucial for axon-guidance in the regenerative process after peripheral nerve injury (PNI). Considering neuregulin-erbB-FAK signaling is an essential pathway participating in the regulation of Sc migration during development, the present study is aimed to examine whether neuregulin would exert its beneficial effects on adult following PNI and further determine the potential changes of downstream pathway engaged in neuro-regeneration by both in vitro and in vivo approaches. Methodology and Principal Findings Cultured RSC96 cells treated with neuregulin were processed for erbB2/3 immunofluorescence and FAK immunoblotings. The potential effects of neuregulin on Sc were assessed by cell adherence, spreading, and migration assays. In order to evaluate the functional significance of neuregulin on neuro-regeneration, the in vivo model of PNI was performed by chronic end-to-side neurorrhaphy (ESN). In vitro studies indicated that after neuregulin incubation, erbB2/3 were not only expressed in cell membranes, but also distributed throughout the cytoplasm and nucleus of RSC96 cells. Activation of erbB2/3 was positively correlated with FAK phosphorylation. Neuregulin also increases Sc adherence, spreading, and migration by 127.2±5.0%, 336.8±3.0%, and 80.0±5.7%, respectively. As for in vivo study, neuregulin significantly accelerates the speed of Sc migration and increases Sc expression in the distal stump of injured nerves. Retrograde labeling and compound muscle action potential recordings (CMAP) also showed that neuregulin successfully facilitates nerve regeneration by eliciting noticeably larger CMAP and promoting quick re-innervation of target muscles. Conclusions As neuregulin successfully improves axo-glial interaction by speeding Sc migration via the erbB2/3-FAK pathway, therapeutic use of neuregulin may thus serve as a promising strategy to facilitate the progress of nerve regeneration after PNI.

Frizzled-10 promotes sensory neuron development in Xenopus embryos

Formation of the vertebrate nervous system requires coordinated cell-cell interactions, intracellular signalling events, gene transcription, and morphogenetic cell movements. Wnt signalling has been involved in regulating a wide variety of biological processes such as embryonic patterning, cell proliferation, cell polarity, motility, and the specification of cell fate. Wnt ligands associate with their receptors, members of the frizzled family (Fz). In Xenopus, five members of the frizzled family are expressed in the early nervous system. We have investigated the role of Xenopus frizzled-10 (Fz10) in neural development. We show that Fz10 is expressed in the dorsal neural ectoderm and neural folds in the region where primary sensory neurons develop. Fz10 mediates canonical Wnt signalling and interacts with Wnt1 and Wnt8 but not Wnt3a as shown in synergy assays. We find that Fz10 is required for the late stages of sensory neuron differentiation. Overexpression of Fz10 in Xenopus leads to an increase in the number of sensory neurons. Loss of Fz10 function using morpholinos inhibits the development of sensory neurons in Xenopus at later stages of neurogenesis and this can be rescued by co-injection of modified Fz10B and beta-catenin. In mouse P19 cells induced by retinoic acid to undergo neural differentiation, overexpression of Xenopus Fz10 leads to an increase in the number of neurons generated while siRNA knockdown of endogenous mouse Fz10 inhibits neurogenesis. Thus we propose Fz10 mediates Wnt1 signalling to determine sensory neural differentiation in Xenopus in vivo and in mouse cell culture.

Proliferative effects of melatonin on Schwann cells: implication for nerve regeneration following peripheral nerve injury

Activation of proliferation of Schwann cells is crucial for axonal guidance and successful nerve regeneration following peripheral nerve injury (PNI). Considering melatonin plays an important role in proliferative regulation of central glial cells, the present study determined whether melatonin can effectively promote Schwann cell proliferation and improve nerve regeneration after PNI. The spontaneous immortalized rat Schwann cell line (RSC 96 cells) was first analyzed by quantitative polymerase chain reaction (QPCR) to detect the potential existence of melatonin receptors. The melatonin receptor‐mediated signaling responsible for proliferation was examined by measuring the phosphorylation of extracellular signal‐regulated kinases (ERK1/2) pathway. The in vivo model of PNI was performed by the end‐to‐side neurorrhaphy. The quantity of Schwann cells as well as the number of re‐innervated motor end plates (MEP) on target muscles was examined to represent the functional recovery of injured nerves. QPCR results indicated that MT1 is the dominant receptor in Schwann cells. Immunoblotting and proliferation assay revealed an enhanced phosphorylation of ERK1/2 and increased number of RSC 96 cells following melatonin administration. Nonselective melatonin receptor antagonist (luzindole) treatment significantly suppressed all the above findings, suggesting that the proliferative effects of melatonin were mediated by a receptor‐dependent pathway. In vivo results corresponded well with in vitro findings in which melatonin effectively increased the amount of proliferated Schwann cells and re‐innervated MEP on target muscles following PNI. As melatonin successfully improves nerve regeneration by promoting Schwann cell proliferation, therapeutic use of melatonin may thus serve as a promising strategy to counteract the PNI‐induced neuronal disability.

β-1,4-Galactosyltransferase III suppresses β1 integrin-mediated invasive phenotypes and negatively correlates with metastasis in colorectal cancer

Metastasis often occurs in colorectal cancer (CRC) patients and is the main difficulty in cancer treatment. The upregulation of poly-N-acetyllactosamine-related glycosylation is found in CRC patients and is associated with progression and metastasis in cancer. β-1,4-Galactosyltransferase III (B4GALT3) is an enzyme responsible for poly-N-acetyllactosamine synthesis, and therefore, we investigated its expression in CRC patients. We found that B4GALT3 negatively correlated with poorly differentiated histology (P < 0.001), advanced stages (P = 0.0052), regional lymph node metastasis (P = 0.0018) and distant metastasis (P = 0.0463) in CRC patients. B4GALT3 overexpression in CRC cells suppressed cell migration, invasion and adhesion, whereas B4GALT3 knockdown enhanced malignant cell phenotypes. The β1 integrin-blocking antibody reversed the B4GALT3-mediated increase in cell invasion. B4GALT3 expression altered glycosylation on the N-glycan of β1 integrin probably through changes in poly-N-acetyllactosamine expression. Furthermore, more activated β1 integrin along with the activation of its downstream signaling transduction were found in B4GALT3 knockdown cells, whereas overexpression of B4GALT3 suppressed the expression of active β1 integrin and inhibited its downstream signaling. Our results suggest that B4GALT3 is negatively associated with CRC metastasis and suppresses cell invasiveness through inhibiting activation of β1 integrin.

Expression of GALNT2 in human extravillous trophoblasts and its suppressive role in trophoblast invasion

Extravillus trophoblast (EVT) invasion plays a critical role in placental development. Integrins bind to extracellular matrix (ECM) proteins to mediate EVT cell adhesion, migration, and invasion. Changes in O-glycans on β1-integrin have been found to regulate cancer cell behavior. We hypothesize that O-glycosyltransferases can regulate EVT invasion through modulating the glycosylation and function of β1-integrin. Here, we found that the GALNT1 and GALNT2 mRNA were highly expressed in HTR8/SVneo and first trimester EVT cells. Immunohistochemstry and immunofluorescence staining showed that GALNT2 was expressed in subpopulations of EVT cells in deciduas, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. The percentage of GALNT2-positive EVT cells increased with gestational ages. Overexpression of GALNT2 in HTR8/SVneo cells significantly enhanced cell-collagen IV adhesion, but suppressed cell migration and invasion. Notably, we found that GALNT2 increased the expression of Tn antigen (GalNAc-Ser/Thr) on β1-integrin as revealed by Vicia Villosa agglutinin (VVA) binding. Furthermore, GALNT2 suppressed the phosphorylation of focal adhesion kinase (FAK), a crucial downstream signaling molecule of β1-integrin. Our findings suggest that GALNT2 is a critical initiating enzyme of O-glycosylation for regulating EVT invasion.

β-1,4-galactosyltransferase III suppresses extravillous trophoblast invasion through modifying β1-integrin glycosylation

INTRODUCTION Glycosylation controls diverse protein functions and regulates various cellular phenotypes. Trophoblast invasion is essential for normal placental development. However, the role of glycosylation in human placenta throughout pregnancy is still unclear. The β-1,4-galactosyltransferase III (B4GALT3) has been found to regulate cancer cell invasion. We therefore investigated the expression of B4GALT3 in placenta and its roles in trophoblast. METHODS B4GALT3 protein expression was examined by quantitative Western blotting analysis in human placentas. For identification of B4GALT3-positive cells in normal human placenta, immunohistochemistry and immunofluorescence methods were used. To investigate effects of B4GALT3 on extravillous trophoblast (EVT)-like cell and primary EVT cells, we analyzed cell growth, adhesion, migration, and invasion in mock and B4GALT3-transfected cell. RESULTS B4GALT3 expression significantly increased in third trimester human placenta. Immunostaining revealed that B4GALT3 expressed in placental villous cytotrophoblast, syncytiotrophoblast, and a subpopulation of EVT cells throughout pregnancy. Interestingly, we found increases in the expression level and percentage of B4GALT3-positive cells in third trimester EVT, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. Overexpression of B4GALT3 in HTR8/SVneo cells and primary trophoblast cells significantly suppressed cell migration. In addition, B4GALT3 suppressed cell invasion, and enhanced cell adhesion to laminin in HTR8/SVneo cells. Notably, we found that B4GALT3 modified glycans on β1-integrin, suppressed focal adhesion kinase (FAK) signaling, and enhanced β1-integrin degradation. DISCUSSION We propose that B4GALT3-mediated glycosylation change not only enhances β1-integrin binding to laminin, but also attenuates β1-integrin stability. Our findings suggest that B4GALT3 is a critical regulator for suppressing EVT invasion in the late stages of pregnancy.