α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells

Acta Pharmacologica Sinica, Jun 2009

Aim: We aimed to investigate the potential modification of previously unrecognized surface glycoprotein(s) by α2,6-sialylation other than by integrins. Methods: The expression of β-galactoside α2,6-sialyltransferase (ST6Gal-I) in the colon cancer cell line HCT116 was reduced by siRNA. The adhesion and Boyden chamber assay were used to detect the variation in cell motility. α2,6-Sialylation proteins were detected with lectin affinity assay. The mRNA expression, protein expression and downstream signaling modulation with siRNA were detected using reverse transcription-polymerase chain reaction, flow cytometry analysis, and Western blot. Results: In HCT116 cells, the knockdown of ST6Gal-I inhibited cell motility, but did not affect cell adhesion. This selectively altered cell migration was caused by the loss of α2,6-sialic acid structures on c-Met. Moreover, STAT3 was dephosphorylated at tyrosine 705 in ST6Gal-I-knockdown (ST6Gal-I-KD) HCT116 cells. Conclusion: c-Met is the substrate of ST6Gal-I. The hyposialylation of c-Met can abolish cell motility in ST6Gal-I-KD HCT116 cells.

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α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells

Acta Pharmacologica Sinica ?2,6-Hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells Jin QIAN 0 Cai-hua ZHU 0 Shuai TANG 1 Ai-jun SHEN 0 Jing AI 1 Jing LI 1 Mei-yu GENG 0 1 Jian DING 0 0 Division of Anti-tumor Pharmacology, Shanghai Institute of Materia Medica , Shanghai 201203 , China 1 Department of Pharmacology, Marine Drug and Food Institute, Ocean University of China , Qingdao 266003 , China Aim: We aimed to investigate the potential modification of previously unrecognized surface glycoprotein(s) by ?2,6-sialylation other than by integrins. Methods: The expression of ?-galactoside ?2,6-sialyltransferase (ST6Gal-I) in the colon cancer cell line HCT116 was reduced by siRNA. The adhesion and Boyden chamber assay were used to detect the variation in cell motility. ?2,6-Sialylation proteins were detected with lectin affinity assay. The mRNA expression, protein expression and downstream signaling modulation with siRNA were detected using reverse transcription-polymerase chain reaction, flow cytometry analysis, and Western blot. Results: In HCT116 cells, the knockdown of ST6Gal-I inhibited cell motility, but did not affect cell adhesion. This selectively altered cell migration was caused by the loss of ?2,6-sialic acid structures on c-Met. Moreover, STAT3 was dephosphorylated at tyrosine 705 in ST6Gal-I-knockdown (ST6Gal-I-KD) HCT116 cells. Conclusion: c-Met is the substrate of ST6Gal-I. The hyposialylation of c-Met can abolish cell motility in ST6Gal-I-KD HCT116 cells. cell motility; c-Met; hyposialylation; ST6Gal-I Introduction Cell surface proteins in mammals are typically elaborated with a complex array of asparagine-linked (N-linked) glycans. Sialic acids are usually found at the non-reducing terminal position of these N-glycans. This terminal sialylation imparts a negative charge at physiological pH values and mediates many biological functions. Altered expression of certain sialic acid types or their linkages is closely associated with cellular adhesion, migration and metastasis in tumor cells[1, 2]. Overexpression of ?-galactoside ?2,6-sialyltransferase (ST6Gal-I) has been observed in numerous types of human tumors, and it results in the increase of N-glycans in the Sia6LacNAc stucture in various cancer cells[3] . Emerging evidences showed that hypersialylation of ?1-integrin stimulated both cell attachment and migration on collagen I and induced variation in cell motility[4, 5]. However, hypersialylation of ?1-integrin could not explain all phenotypic events. We therefore hypothesized that ST6Gal-I could modify other surface glycoprotein(s), which might directly or in synergy with integrins be responsible for the biological behavior. In the present study, we aimed to investigate the potential modification of previously unrecognized surface glycoprotein(s) by ST6Gal-I. The expression of ST6Gal-I in the human HCT116 colon carcinoma cell line was transiently knocked down with siRNA. Finally, we demonstrated a functional role for ST6Gal-I-driven terminal sialylation of c-Met glycoprotein in colon cancer progression, which might be distinct from integrin-dependent cancer progression. Materials and methods Cell lines and culture conditions The colon cancer adenocarcinoma cell line, HCT116, was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were propagated and maintained in McCoy?s 5A Medium (Sigma, St Louis, MO, USA) containing 100 U/mL penicillin and supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA). Cells were maintained at 37 ?C under a humidified 95% and 5% (v/v) mixture of air and CO2. Short interfering (si)RNA transfection siRNA duplexes (D3) 5?-AACTCTCAGTTGGTTACCACA-3? specific for the human ST6Gal-I gene sequences, as well as control nontargeting siRNA (NC), were purchased from npg 1040 Shanghai Genepharma Co Ltd (Shanghai, China). Briefly, HCT116 cells were grown to 75% confluence, exposed to siRNA (100 nmol/L) for 6 h in the presence of oligofectamine (Invitrogen, Carlsbad, CA, USA), and then incubated at 37?C for 24 h before use in assays. RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from transfected cells using TRIzol (Invitrogen) after cells had reached about 90% confluence. After RT, cDNA was amplified by PCR using Taq DNA polymerase, deoxynucleoside triphosphates and the indicated primers, as described previously[6], and 30 cycles of amplification were performed. The PCR products (10 ?L) were analyzed by electrophoresis on 2% agarose gels. After being stained with ethidium bromide, gel images were obtained using Genesnap Version 6.0026 software (Syngene, Cambridge, England). ST6Gal-I activity detection The activity of ST6Gal-I was determined by an enzyme-linkedimmunosorbent assay (ELISA) using asialofetuin (Sigma) precoated plates. Equivalent amounts of cell lysates were loaded and cytidine-5?-monophospho-N-acetylneuraminic acid sodium salt (CMP-NeuAC, Sigma) was then added to initiate the reaction. After being washed and blocked, the sialylated fetuin was bound with SNA-biotin (Vector Lab, Burlingame, CA, USA), and probed with streptavidin-horseradish peroxidase. Finally, 100 ?L of solution (0.03% H2O2, 2 mg/mL o-phenylenediamine in citrate buffer 0.1 mol/L, pH 5.5) was added and the reaction was terminated with H2SO4. The absorbance at 492 nm was measured using a multiwell spectrophotometer (VERSAmax, Molecular Devices, Union City, CA, USA). Fluorescence activated cell sorting (FACS) analysis For analysis of the cell surface ?2,6-sialic acid structure and c-Met protein, adherent HCT116 cells were trypsinized and placed in FACS blocking buffer at a concentration of 5?105 cells/mL. The cells were then incubated with 2 ?g SNA-biotin or 0.5 ?g c-Met antibody (R&D, Minneapolis, MN, USA) for 30 min on ice, washed twice in blocking buffer, and then exposed to 0.25 ?g R-phycoerythrin (RPE)-conjugated streptavidin for an additional 30 min. Labeled samples were analyzed by flow cytometry. Cell adhesion assay Adhesion assays were performed in 96-well plates pre-coated with collagen (10 ?g/mL), fibronectin (10 ?g/mL) or laminin (20 ?g/mL) (BD, San Jose, CA, USA). After being blocked with 1% bovine serum albumin, 0.4?105 cells were seeded into each well and allowed to adhere at 37 ?C for 1 h. Non-adherent cells were rinsed with phosphate-buffered saline (PBS); the remaining cells were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet for 15 min. After being rinsed with water, the remaining cells were solubilized by acetic acid. Absorbance spectroscopy (595 nm) was used to quantify the number of substrate-adherent cells. Migration assay Transwell Boyden chambers (Costar, Bethesda, MD, USA) were coated with gelatin. After being soaked with serum-free 5A medium, 2?105 transfected cells suspended in 5A containing 2% FBS were loaded into the upper well. The lower compartment was filled with culture medium alone or medium containing 100 ng hepatocyte growth factor (HGF, R&D) and then incubated at 37 ?C for 30 or 48 h. After the non-migratory cells were removed, the migrated cells were fixed and stained as previously described and counted. Preparation of total cell extracts and immunoblot analysis After transfection with siRNA for 24 h, cells were harvested and resuspended in RIPA lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% SDS, 0.5% deoxycholic acid, 0.02% sodium azide, 1% NP-40, 2.0 ?g/mL aprotinin, 1 mmol/L phenylmethylsulfonylfluoride). The effects of HGF stimulation were detected by serum starving transfected cells for 24 h and then stimulating with 50 ng HGF for 20 min. Equivalent amounts of proteins were used for Tyr1349-MET, Erk1/2, phosphorylated-Erk1/2, AKT, phosphorylated-AKT, STAT3, Tyr705-STAT3, Ser727-STAT3 (Cell Signaling Technology, Beverly, MA, USA) and/or ?-subunit-specific antibody for MET (Santa Cruz Biotech, Santa Cruz, CA, USA) immunoblot detection. Lectin affinity assays Lysates were centrifuged at 10 000?g for 15 min at 4 ?C. One milligram of supernatant was incubated for 4 h at 4 ?C with 6 ?g SNA. Streptavidin-agarose beads (Sigma) were then added and incubated for an additional 4 h at 4 ?C with rotation. After being briefly centrifuged and washed, precipitated proteins were released from the bead complexes by boiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and analyzed directly by SDSPAGE and immunoblotting. FACS analysis For analysis of the cell surface ?2,3-sialic acid structure, adherent HCT116 cells were trypsinized with trypsin-EDTA using standard methods and placed in FACS blocking buffer (0.15 mol/L PBS with 2% BSA) at a concentration of 5?105 cells/mL. The cells were then incubated with 2 ?g MAAbiotin (Vector Lab, Burlingame, CA) for 30 min on ice, washed twice in blocking buffer, and then exposed to 0.25 ?g RPEconjugated streptavidin for an additional 30 min. Labeled samples were analyzed by flow cytometry. 5-bromo-2?-deoxyuridine (BrdU) assay DNA synthesis was monitored by measuring incorporation of the artificial thymidine nucleotide analog BrdU (Sigma, St Louis, MO, USA) into newly synthesized DNA. Cells (1.5?105 per well) were cultured in 6-well plates and transfected with siRNAs. Forty-eight hours after transfection, cells were refreshed with complete medium containing 100 ?mol/L BrdU and incubated for an additional 30 min for BrdU incorporation. After BrdU incubation, cells were washed three times with PBS and fixed in 4% paraformaldehydePBS for 20 min. Next, the fixed cells were permeabilized with 0.5% Triton X-100-PBS. After partial denaturation of the DNA with 2 mol/L HCl, cells were incubated with anti-BrdU mouse monoclonal antibody (Santa Cruz Biotechnology, CA), at a 1:50 dilution for 1 h. Cells were then washed with PBS three times and incubated with Alexa Fluor 633 anti-mouse antibody (Invitrogen, Carlsbad, CA, USA) at a 1:200 dilution for 1 h. After three PBS washes, cells were counterstained with 4?,6?-diamidino-2-phenylindole for 5 min and analyzed using an Olympus DP70 digital microscope camera. Quantitative analysis of immunofluorescence data was carried out with Image-Pro Plus software. Results Knockdown of ST6Gal-I reduced endogenous ST6Gal-I expression in HCT116 cells Cell surface sialylation of the metastasizing HCT116 cell line correlates with in vivo tumorigenicity[2], and overexpression of ST6Gal-I has been implicated in increased cell motility. Here, we sought to determine the effect of in vitro knockdown of ST6Gal-I expression on the motility of HCT116 cells. We transiently transfected ST6Gal-I-targeting siRNA into HCT116 cells (D3) and verified that ST6Gal-I mRNA expression was reduced by RT-PCR detection in a time-dependent manner (Figure 1A). The activity of ST6Gal-I in siRNA transfected HCT116 cells (D3) was compared with that in parental HCT116 cells (P) and HCT116 cells transfected with nonspecific siRNA (NC) by ELISA assay. After interference with ST6Gal-I-targeting siRNA, the ?2,6-sialic acid decoration was significantly decreased in D3 cells (Figure 1B). The ?2,6-sialic acid structures on the cell surface were evaluated by FACS analysis. As expected, the ?2,6-sialic acid structures on the D3 cell surface were remarkably reduced compared with those in P and NC cells (Figure 1C), whereas the ?2,3-sialic acid structures were not affected (Figure S1). These results indicated that siRNA effectively reduced the expression of ST6Gal-I. npg Decreased expression of ST6Gal-I reduces cell migration but not cell adhesion To determine the effect of cell surface ?2,6-sialylation on cell motility, we evaluated cell migration using a modified Boyden c-Met is hyposialylated in ST6Gal-I-KD HCT116 cells chamber assay with FBS as a chemoattractant. The migration It has been postulated that changes in sialic acid structure of D3 cells was significantly less than that of P or NC cells (Fig- on membrane glycoconjugates can modulate the adhesion ure 1D). The growth kinetics study showed that the reduc- of cancer cells to ECM components in an integrin-dependent tion in ST6Gal-I had no effect on cell proliferation (Figure S2). manner[7?10]. Because we did not find any change in ST6GalThese results indicate that the ST6Gal-I regulated cell motility I-KD HCT116 cell adhesion, we suggest that integrins alone but not cell growth. cannot fully explain this observation. Sialylation of membrane We next examined the effects of ST6Gal-I siRNA on cell glycoproteins other than integrins might be responsible for the adhesion, specifically to three main extracellular matrix (ECM) ST6Gal-I-dependent motility phenotype in HCT116 cells. To components: laminin, fibronectin, and collagen. The ability of identify the related membrane glycoproteins in the regulation D3 cells to attach to these three components was similar to that of P and NC cells (Figure 1E), suggesting that downregulation of ST6Gal-I expression had no effect on cell adhesion. npg of cell motility, we precipitated ?2,6-sialylated glycoproteins using SNA and then examined the glycoproteins of interest, c-Met and CXCR4, by immunoblotting. Remarkably, c-Met was clearly present in SNA precipitates prepared from cells with normal ST6Gal-I expression (P and NC), whereas little c-Met was present in ST6Gal-I-KD HCT116 cells (D3) (Figure 2A). In contrast, CXCR4 was not co-precipitated by SNA (data not shown). To further examine the possibility that the decreased cell motility of ST6Gal-I-KD HCT116 cells was caused by the abolition of normal c-Met function, we evaluated HGF-stimulated migration using Boyden chamber assays. In D3 cells, HGF stimulation induced less migration and also eliminated the ability to trigger the development of multicellular-branched structures compared with P and NC cells (Figure 2B). These results indicate that down-regulation of ST6Gal-I expression resulted in c-Met hyposialylation and inhibition of HGFinduced motility of HCT116 cells. Insufficient terminal ?2,6-sialylation abolishes c-Met maturation To elucidate how insufficient terminal ?2,6-sialylation determines the fate of the c-Met molecule, we examined c-Met mRNA and protein expression in ST6Gal-I-KD HCT116 cells. Reduced terminal ?2,6-sialylation was associated with a decrease in c-Met expression on the surface of D3 cells relative to P or NC cells (Figure 3A). However, there were no differences in mRNA expression among the three tested cell lines (Figure 3B). These findings indicate that ST6Gal-I affected c-Met function at the post-translational level, rather than at the transcriptional or translational level. The endoplasmic reticulum (ER)-resident precursor form of c-Met is glycosylated and cleaved into two Golgi-processed mature subunits (? and ?) that are further linked via disulfide bonds into a functional heterodimer on the cell surface. We therefore investigated whether the terminal deficiency in ?2,6linkage disrupted c-Met maturation in ST6Gal-I-KD HCT116 cells. Using ?-subunit-specific antibodies, we found that mature ?-subunit was significantly decreased in D3 cells compared with P or NC cells. Consistently, a reduction in phosphorylated c-Met was noted, mimicking the protein expression profile (Figure 3C). Tyr705, but not Ser727 phosphorylation of STAT3 responds to hyposialylation in HCT116 cells We investigated three key downstream targets of c-Met in these transfected HCT116 cells under normal culture conditions. Phosphorylation of STAT3 was down-regulated at Tyr705, rather than Ser727, in ST6Gal-I-null cells, whereas the total level of STAT3 remained unchanged. The other two downstream effectors, Erk1/2 and AKT, were not affected (Figure 4A). Moreover, there was no significant modulation on any of the downstream signaling pathways when cells were starved and stimulated with HGF alone (Figure 4B). These results indicate that insufficient terminal ?2,6-sialylation of c-Met or other unidentified glycoproteins directly or indirectly regulate STAT3 Tyr705 dephosphorylation. Discussion Increased levels of ST6Gal-I and ?2,6-sialic acid have been observed in various cancer cells and are associated with a poor prognosis in cancer progression[11]. In vitro cell culture studies suggest that ST6Gal-I up-regulation may contribute to metastasis by regulating invasiveness and cell motility. The role of the integrin family members has been emphasized in this context by modifying adhesion to ECM components. However, some studies have reported conflicting results. For example, terminal sialylation reduces attachment to ECM glycoproteins in melanoma[7] and in myeloid cells[8], but enhances adhesion in erythroleukemia K562 cells[9] and colon epithelial SW948 npg cells[10]. These observations support the possibility that integrins are not the only relevant targets of ST6Gal-I-mediated sialylation in cancer cell adhesion and suggest that other surface glycoproteins may also be involved. Using the siRNA technique in combination with an SNA lectin-blotting approach, we found, for the first time, that ST6Gal-I knock-down specifically abolished HCT116 cell motility via terminal sialylation of c-Met. Despite numerous reports on the glycoprotein nature of c-Met, the potential glycosylation pattern of N-linked carbohydrates on c-Met has not been extensively studied. In our study, ST6Gal-I deficiency npg caused a reduction in ?2,6-sialylation of c-Met, supporting the substrate of ST6Gal-I and that its function is regulated by idea that c-Met is the substrate of ST6Gal-I. modification of its ?2,6-sialic terminal structure. We also Under physiological conditions, c-Met activation is a rela- found that elimination of a single terminal sialic acid structure tively transient event, whereas in tumor cells, c-Met is often could affect signaling events in HCT116 cells. These findings constitutively activated[12]. c-Met activation in human tumor suggest that therapies targeting toward the ST6Gal-I glycosylcells can be initiated through various mechanisms, including transferase gene may be beneficial, either directly or through overexpression, structural alterations, and receptor deregula- synergistic interactions, in treating malignant tumors harbortion. The results of the current study suggest that the c-Met ing constitutively activated c-Met. hyposialylation is likely to result in long-lasting phenotypic changes. In contrast to the rapid and transient regulatory Acknowledgments dynamic characteristics of mature forms of c-Met, the altered This work was supported by the National Basic Research Proglycosylation patterns typical of immature c-Met prevent its gram Grant (No 2003CB716400) of China, the Natural Science efficient trafficking and thus limit its localization to the cell Foundation of China for Distinguished Young Scholars (No surface. In this context, ST6Gal-I-driven c-Met activation 30725046) and the Natural Science Foundation of China for could account for the long-term changes in c-Met-mediated Innovation Research Group (No 30721005). cell motility that are characteristic of neoplastic cells. Similar to many other growth factor receptor tyrosine Author contribution kinases, c-Met exerts its oncogenic potential through deregu- Prof Mei-yu GENG, Prof Jian DING and Prof Jing LI designed lating the activation of a number of protein phosphorylation- the research. Jin QIAN performed the research, analyzed dependent signaling cascades[13]. These include the Erk/ data and wrote the paper. Cai-hua ZHU, Shuai TANG, Ai-jun MAPK and PI3K pathways, which are important for cell SHEN, and Jing AI contributed to the research. adhesion, proliferation, and survival. In the present study, ST6Gal-I null failed to induce adhesion or proliferation. References Consistent with this observation, the levels of both total and 1 Collard JG, Schijven JF, Bikker A, La Riviere G, Bolscher JG, Roos E. phosphorylated forms of Erk1/2 and AKT were essentially Cell surface sialic acid and the invasive and metastatic potential of unchanged following the ST6Gal-I knockdown. Further stud- T-cell hybridomas. Cancer Res 1986; 46: 3521?7. ies are needed to clarify the biological basis for this lack of 2 Morgenthaler J, Kemmner W, Brossmer R. Sialic acid dependent cell effect. adhesion to collagen IV correlates with in vivo tumorigenicity of the In addition to the Erk/MAPK and PI3K pathways, the acti- hBuiomchaenmcoBlioonphcyasrcRinesomCoamsmubulnin1es99H0C;T117116: ,8H60C?T161.6a and HCT116b. vation of c-Met can also induce cell invasion and migration 3 Wang PH, Li YF, Juang CM, Lee YR, Chao HT, Tsai YC, et al. Altered through a complex network of adaptors and transducers. One mRNA expression of sialyltransferase in squamous cell carcinomas of of these, the transcription factor STAT3, is a key downstream the cervix. Gynecol Oncol 2001; 83: 121?7. target of c-Met signaling[14]. STAT3 is constitutively activated 4 Seales EC, Jurado GA, Singhal A, Bellis SL. Ras oncogene directs in a number of human tumors and possesses oncogenic poten- expression of a differentially sialylated, functionally altered beta1 tial and anti-apoptotic activities. The precise roles of STAT3 integrin. Oncogene 2003; 22: 7137?45. in c-Met signal transduction are not completely understood. 5 Seales EC, Jurado GA, Brunson BA, Wakefield JK, Frost AR, Bellis Some reports suggested that STAT3 bound directly to c-Met[14] SL. Hypersialylation of beta1 integrins, observed in colon adenoand led to c-Met-dependent formation of branched tubules. carcinoma, may contribute to cancer progression by up-regulating cell Others hold the view that c-Met activates STAT3 indirectly, motility. Cancer Res 2005; 65: 4645?52. leading to anchorage-independent growth[15]. Regardless 6 LsiianlySla,tKioenmamffencetrs Wad,hGersigiounll oSf, bSrcehalsatgcaPrMci.noCmeall cseullrsf.acEexpalpChelal R2e,6s of the mechanism, activation by phosphorylation at Tyr705 2002; 276: 101?10. induces STAT3 dimerization, nuclear translocation and DNA 7 Pochec E, Litynska A, Amoresano A, Casbarra A. Glycosylation profile binding[16], whereas phosphorylation at Ser727 is responsible of integrin alpha 3 beta 1 changes with melanoma progression. for STAT3 transcriptional activation[17]. In the current study, Biochim Biophys Acta 2003; 1643: 113?23. dephosphorylation at Tyr705, but not at Ser727, was observed 8 Semel AC, Seales EC, Singhal A, Eklund EA, Colley KJ, Bellis SL. in ST6Gal-I-KD HCT116 cells upon FBS stimulation. By con- Hyposialylation of integrins stimulates the activity of myeloid fibrotrast, in D3 cells, HGF stimulation obviously affects c-Met nectin receptors. J Biol Chem 2002; 277: 32830?6. phosphorylation but has no effect on the phosphorylation 9 Symington BE, Symington FW, Rohrschneider LR. Phorbol ester status of STAT3 at either Tyr705 or Ser727. These results sug- induces increased expression, altered glycosylation, and reduced gest that ?2,6-hyposialylated c-Met could abolish c-Met matu- adhesion of K562 erythroleukemia cell fibronectin receptors. J Biol rsatitmiounlia.nTdhedeosbtsreoryveitds dmeoplheocusplahrosreynlastiitoivnitayt Ttoyre7n0v5iriso nlimkeelnyttaol 10 CcChhhaeirnmigceo1sl9o8inM9d;,u2cM6e4ad:lab1gy3o2el5ixnp8ir?eN6s,6sBi.oonnofifgblieotlai-Sg,alDaactlol?sOi dlieo aFl.phPah2e,6nostiyaplyilcbe a consequence of ?2,6-hyposialylation of all target proteins, transferase I in the human colon cancer cell line SW948. Glycobiology rather than being unique to c-Met. The potential involvement 2006; 16: 146?54. of other glycoproteins merits further investigation. 11 Lise M, Belluco C, Perera SP, Patel R, Thomas P, Ganguly A. Clinical In conclusion, we have shown that c-Met is an additional correlations of alpha2,6-sialyltransferase expression in colorectal cancer patients . Hybridoma 2000 ; 19 : 281 - 6 . 12 Takayama H, LaRochelle WJ , Sharp R , Otsuka T , Kriebel P , Anver M , Natl Acad Sci USA 1997 ; 94 : 701 - 6 . 13 Blume-Jensen P , Hunter T. Oncogenic kinase signalling . Nature 2001 ; 411 : 355 - 65 . 14 Cramer A , Kleiner S , Westermann M , Meissner A , Lange A , Friedrich K. cell behavior by signaling through transcription factor STAT3 . J Cell Biochem 2005 ; 95 : 805 - 16 . 15 Zhang YW , Wang LM , Jove R , Vande Woude GF. Requirement of Stat3 signaling for HGF/SF-Met mediated tumorigenesis . Oncogene 2002 ; 21 : 217 - 26 . 16 Darnell JE Jr, Kerr IM , Stark GR . Jak-STAT pathways and transcriptional proteins. Science 1994 ; 264 : 1415 - 21 . 17 Patten I , Placzek M. Opponent activities of Shh and BMP signaling during floor plate induction in vivo . Curr Biol 2002 ; 12 : 47 - 52 .


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Jin Qian, Cai-hua Zhu, Shuai Tang, Ai-jun Shen, Jing Ai, Jing Li, Mei-yu Geng, Jian Ding. α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells, Acta Pharmacologica Sinica, 2009, 1039-1045, DOI: 10.1038/aps.2009.84