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Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated... Background: Previously, we found that b-galactoside a2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi- localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin b1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration. Methods: We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin b1. After ionizing radiation, migration of cells was measured by in vitro migration assay. a2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an a2, 6 sialyltransferase sandwich ELISA. Result: We found that ST6Gal I was cleaved by BACE1 (b-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non- cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin b1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin b1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by BACE 1 inhibited integrin b1 sialylation and migration by Golgi-anchored form of ST6Gal I. Conclusions: Our results suggest that soluble ST6Gal I, possibly in cooperation with the Golgi-bound form, may participate in cancer progression and metastasis prior to being secreted from cancer cells. Keywords: BACE1, Migration, Radiation, ST6Gal I * Correspondence: ygko@korea.ac.kr; yslee0425@ewha.ac.kr † Contributed equally College of Life Sciences and Biotechnology, Korea University, 1, 5-ka, Anamdong, Sungbuk-gu, Seoul 136-701, South Korea College of Pharmacy & Division of Life & Pharmaceutical Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul 120-750, South Korea Full list of author information is available at the end of the article © 2012 Lee et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lee et al. Radiation Oncology 2012, 7:47 Page 2 of 10 http://www.ro-journal.com/content/7/1/47 forms of glycosyltransferases exist in the plasma of Background patients with certain diseases, and can sometimes be ST6Gal I (b galactoside a2,6 sialyltransferase, CMP- used as biomarkers for these diseases [30-33]. NeuAc: Galb (1,4) GlcNAc: a2,6 sialyltransferase) is an In the present study, we examined IR-induced clea- important glycosyltransferase that adds a sialic acid resi- vage and solubilization of ST6Gal I, which is released due to the terminal position on N-linked oligosacchar- into the cell culture media of colon cancer cell lines, ides [1,2]. It is localized in the Golgi apparatus in a and sought to identify the protease involved in cleaving membrane-anchored form and is cleaved into a secre- ST6Gal I after exposure to IR. We found that BACE1 tary protein by cathepsin-like proteases [3]. Recent stu- could be the secretase responsible for IR-induced clea- dies and clinical reports have emphasized the vage of ST6Gal I, and showed that BACE1 mediated importance of ST6Gal I in cancer progression and cleavage of ST6Gal I decreased ST6Gal I -mediated can- metastasis. ST6Gal I is up-regulated in colon adenocar- cer cell migration. Additionally, the soluble form of cinoma and its expression is positively associated with ST6Gal I possesses sialyltransferase enzymatic activity, tumor cell migration and invasion [4-6]. Specifically, but unlike the Golgi-associated form, did not sialylate patients with metastasizing tumors have high levels of integrin b1 to a significant degree, suggesting that solu- ST6Gal I in their serum, and serum levels of ST6Gal I ble ST6Gal I, possibly in cooperation with the Golgi- are correlated with the progression of colorectal carci- bound form, may participate in cancer progression and nomas and cancer metastasis [7-13]. However, a possible metastasis before being secreted from cancer cells, inde- biological role of ST6Gal I in the plasma has not been pendent of intergrin b1-sialylation. reported. Metastasis represents an obligatory step in cancer Materials and methods progression. A variety of molecules contribute to can- Cell culture cer progression and metastasis [14], and many of the SW480 and SW48 human colorectal carcinoma cells factors that function in tumor metastasis are glycopro- were growninRPMImedia supplemented with heat- teins [15-17]. It has been previously demonstrated that inactivated 10% fetal bovine serum and antibiotics. integrin b1 is a major substrate of ST6Gal I [4,18]. In CT26 mouse colorectal carcinoma cells were cultured in colon epithelial cells, oncogenic Ras has been shown to Dulbecco’s modified Eagle’s medium (DMEM) supple- up-regulate ST6Gal I expression, leading to increased mented with heat-inactivated 10% fetal bovine serum a-2,6 sialylation of b1 integrin [19]. Hypersialylation of and antibiotics. integrin b1 augments colon cancer metastasis by alter- ing cellular preference for a certain extracellular matrix milieu as well as by stimulating cell migration. Integ- Plasmids and transfection For transient transfection experiments, expression con- rins also regulate cellular functions, including survival, structs of C-terminally Flag-tagged wild-type (WT; p3 × proliferation and cell spreading, through the function Flag-ST6Gal I) ST6Gal I and N-terminally deleted (HA- of signaling molecules co-localized to the focal adhe- tagged) ST6Gal I (ST6Gal I ΔN) were created as sion complex [20,21]. We have previously demon- described previously [23]. An N-terminally Flag-tagged strated that exposure to ionizing radiation (IR) double-mutant, ST6Gal I (L37A/K40A) was created by increases the expression of ST6Gal I as well as the site-directed mutagenesis [34]. Predesigned small inter- level of sialylated glycoprotein. Sialylation of integrin fering RNA (siRNA) for ST6Gal I was purchased from b1 by exposure of cells to IR increases the adhesion Dharmacon (Lafayette, CO, USA). BACE-1 was targeted and migration of colon cancer cells through integrin with the siRNA duplex 5’-AGA UCC UGU CCA UUG b1-mediated cellular signaling. Therefore, integrin b1 AU CUC CAC CC-3 ’ and 5 ’-GGG UGG AGA UCA sialylation and the subsequent activation of p130CAS, AUG GAC AGG AUC U-3’ [35]. A BACE-1 expression paxillin, and AKT signaling may be one of the plasmid (pcDNA3.1/BACE-1) was generously provided mechanisms involved in IR-mediated-radioresistance by Dr. Sul-Hee Chung (Inje University, Pusan, Korea) and cancer metastasis [22-26]. [36]. Cells were transfected with plasmids using Lipofec- b-site amyloid precursor protein-cleaving enzyme tAMINE 2000 (Invitrogen, Carlsbad, CA, USA) as (BACE) is a membrane-bound aspartic protease that described by the manufacturer. cleaves the amyloid precursor protein (APP) in the pathogenesis of Alzheimer’s disease [27,28]. Importantly, Irradiation BACE has been identified as a protease responsible for Cells were exposed to g-irradiation using a Cs g-ray the cleavage and secretion of Golgi-resident ST6Gal I [29]. The mechanisms underlying cleavage are compli- source (Atomic Energy of Canada, Mississauga, ON, cated, and have not been well characterized. Soluble Canada) at a dose rate of 3.81 Gy/min. Lee et al. Radiation Oncology 2012, 7:47 Page 3 of 10 http://www.ro-journal.com/content/7/1/47 Immunoblot, lectin affinity assay, and and then analyzed for soluble ST6Gal I levels using an immunoprecipitation a2,6 sialyltransferase sandwich enzyme-linked immuno- Protein levels were detected using the following commer- sorbent assay (ELISA) kit (IBL, Japan), according to the cial antibodies: anti-integrin b1 (BD Biosciences, Franklin manufacturer’s protocol. Lakes, NJ, USA); anti-phospho-p130CAS, anti-phospho- Src, anti-HA, and anti-Myc (Cell Signaling Technology, Statistical analysis Danvers, MA, USA); anti-Flag (Sigma); and anti-ST6Gal I Data are expressed as means ± standard deviations and anti-BACE-1 (IBL, Japan). For detection of sialylated (SDs). Statistical significance was determined using Stu- proteins, cell lysates were incubated with biotinylated SNA dent’s t-test for comparisons between two means. The (Sambucus Nigra lectin; Vector Laboratories, Burlingame, null hypothesis was rejected in cases where p-values CA, USA), and protein-lectin complexes were precipitated were < 0.05. with avidin-coated protein A-agarose (Sigma). Results Flow cytometry Inhibition of ST6Gal I cleavage by knockdown of BACE1 Cells were detached with trypsin/EDTA at the indicated increases sialylation of cell surface protein times and stained with fluorescein isothiocyanate In our previous study, we found that IR exposure (FITC)-conjugated SNA (FITC-SNA) or Maackia amur- induces ST6Gal I cleavage and produces a soluble form ensis (FITC-MAA) lectin (Vector Laboratories, Burlin- of ST6Gal I in SW480 colon cancer cells [23]. To sup- game, CA, USA) for detection of a2,6 and a2,3 port the result of our prior work, we next examined the sialylation, respectively. After staining, fluorescence expression of soluble ST6 Gal I in SW48 cells (ST6 Gal -/- intensity was analyzed by fluorescence-activated cell I ) by transiently transfecting these cells with expres- sorting (FACS). sion vectors for Golgi-anchored (N-terminally Flag- tagged and C-terminally Myc-tagged) of ST6Gal I and Reverse transcription-polymerase chain reaction (RT-PCR) exposing them to IR. As shown in Figure 1A, both the Total RNA was isolated with TRI reagents (Molecular Golgi-anchored and soluble forms of ST6Gal I were Research Center), and reverse transcription was done expressed in SW48 colon cancer cells (Figure 1A). using Omniscript transcriptase (Qiagen) under the fol- Because it had previously been demonstrated that sialyl- lowing thermocycling conditions: 30 cycles of 95°C for 5 transferases are cleaved by cathepsin-like protease or min (denaturing), 60°C for 30 s (annealing), and 72°C BACE [3,29,34,37], we sought to identify the target pro- for 30 seconds (extension). The primer sequences used tease(s) responsible for cleavage of ST6Gal I using var- were as follows: human BACE1, 5’-GGT GGA GAT ious protease inhibitors in SW480 vector-control and CAA TGG ACA GG-3’ (sense) and 5’-CGT GGA TGA ST6Gal I-overexpressing cells. Inhibitors of b-secretase CTG TGA GAT GG-3’ (antisense); mouse Bace1, 5’- and g-secretase increased the protein levels of the GCAGAC CCACAT TCCGAA CA-3’ (sense) and 5’- uncleaved, proform of ST6Gal I, whereas cathepsin B GCC ACT GTC CAC GAT GCT CTT-3’ (anti-sense); and L (CTS) inhibitors did not (Figure 1B). In addition, and GAPDH, 5’-CAT GGA GAA GGC TGG GGC TCA a2, 6 sialylation level at the cell surface, detected by TTT-3’ (sense) and 5 ’-CGC CAG TAG AGG CAG FITC-conjugated SNA, was also increased by treatment GGA TGA TGT-3’ (antisense). with b-secretase or g-secretase, but not CTS, inhibitors. In contrast, a2, 3 sialylation at the cell surface, detected In vitro migration assay by FITC-MAA, was not altered by these inhibitors (Fig- Cell migration assays were performed using a Boyden ure 1C), suggesting that b-secretase or g-secretase might chamber as previously described [24]. Cells were plated be responsible for the cleavage of ST6Gal I in SW480 on the upper side of a collagen treated, polycarbonate colon cancer cells. Next, because BACE1 is reported to membrane separating two chambers of 6.5-mm Trans- be the protease responsible for the cleavage of ST6Gal 1 well culture plates (Costar, Corning, NY, USA). After 24 [34,35], we tested the effects of siRNA-mediated BACE1 hours, cells on the upper face of the membrane were knockdown. We observed a concomitant increase in the scraped using a cotton swab and cells that had migrated level of non-cleaved ST6Gal I protein and sialylation of to the lower face of the membrane were stained with integrin b1, a substrate of ST6Gal I (Figure 1D). More- DiffQuick (Baxter Scientific, Deerfield, IL, USA) Wright- over, knockdown of BACE1 expression augmented the Giemsa Solution. sialylationonthesurfaceofthese cells. Interestingly, BACE1 overexpression did not specifically inhibit cell Soluble ST6Gal I ELISA surface sialylation (and may have slightly increased it) Cell culture media were concentrated using Centricon compared with control levels (Figure 1E), suggesting centrifugal filter devices (Millipore, Billerica, MA, USA) that BACE1-mediated cleavage of ST6Gal I only slightly Lee et al. Radiation Oncology 2012, 7:47 Page 4 of 10 http://www.ro-journal.com/content/7/1/47 Figure 1 Inhibition of BACE1 stabilizes ST6Gal I and increases cell surface sialylation. A. SW48 (ST6Gal I-negative) colon cancer cells were transiently transfected with an N-terminally Flag-tagged ST6Gal I expression plasmid, and 24 hours later, cells were exposed to IR (10 Gy). Cell lysates were assessed for expression of ST6Gal I by immunoblotting with an ST6Gal I antibody. B. SW480 vector-control cells and cells stably expressing ST6Gal I were treated with inhibitors of b-secretase, g-secretase, or cathepsin B&L at a concentration of 20 μM. After incubating for 24 hours, cells were harvested and ST6Gal I levels were analyzed by immunoblotting with an anti-ST6Gal I antibody. C. Cell surface a2, 6 sialylation (SNA-FITC) and a2, 3 sialylation (MAA-FITC) were analyzed by FACS after 24 hours of treatment with 20 μM b-secretase or g-secretase inhibitors. D. SW480 cells were transfected with siRNA against BACE1, and the levels of BACE1, ST6Gal I, and integrin b1 protein were assessed by immunoblotting. Sialylation of integrin b1 was assayed by SNA lectin affinity assay. E. SW480 vector-control cells and cells stably expressing ST6Gal I were treated with Si-BACE1 or transfected with a BACE1 expression plasmid. Then, a2, 6 sialylation (SNA-FITC) of the cell surface was analyzed by FACS. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). shows the sialylation potential of ST6Gal I. Taken glycoproteins were barely sialylated compared to WT or together, these results suggest that BACE1 may be L37A/K40A (Figure 2C). These results show that the responsible for cleavage of ST6Gal I. non-cleavable form of ST6Gal I has enzymatic activity and increases a2, 6 sialylation at the cell surface to the The non-cleavable, double-mutant ST6Gal I (L37A/K40A) same extent as WT. However, it was suggested that the increases a2, 6 sialylation at the cell surface sialylation targets of the soluble form of ST6Gal I were It was previously demonstrated that replacement of not cell surface proteins. This result may be due to the ST6Gal I residues Leu37 and Lys40 with alanine signifi- fact that there isn’t much CMP-sialic acid substrate on cantly decreases BACE1-mediated cleavage of ST6Gal I the outside of the cell. [34]. Therefore, we prepared a N-terminally Flag-tagged and C-terminally myc-tagged, non-cleavable ST6Gal I Non-cleaved form ST6Gal I shows integrin b1 dependent (L37A/K40A)mutantandan HA-tagged, N-terminally cell migration truncated soluble form of ST6Gal I (ΔN, amino acids Integrin b1, aknownsubstrateofST6GalI[4,38], 43-403) lacking the protease-specific recognition motif together with associated adaptor molecules, such as (Figure 2A). We then transiently transfected cells with p130CAS, plays a role in promoting the migration of ST6Gal I WT, L37A/K40A or ΔN constructs, and exam- cancer cells [39-41]. We previously reported that integ- ined ST6Gal I protein expression by immunoblotting rin b1 sialylation and its associated cancer cell migra- (Figure 2A) and measured ST6Gal I enzymatic activity tion-related signaling activity are increased by IR- and sialylation of cell surface proteins 24 hours later. induced ST6Gal I [22,23,26]. The phenomena described We found that the enzymatic activity of both the non- above prompted us to compare the action of WT and cleavable L37A/K40A mutant and the ΔNsoluble form non-cleavable L37A/K40A on integrin b1 sialylation, was similar to that of WT (Figure 2B). In addition, integrin b1-mediated signaling, and cell migration. As L37A/K40A increased cell surface sialylation to a similar shown in Figure 3A, transfection with L37A/K40A extent as WT. However, in the case of ΔN, which also increased the Tyr410-phosphorylated (active) form of Y410 had enzymatic catalytic activity, cell surface p130CAS (p-p130CAS ) and sialylation of integrin b1 Lee et al. Radiation Oncology 2012, 7:47 Page 5 of 10 http://www.ro-journal.com/content/7/1/47 Figure 2 Construction of ST6Gal I expression plasmids and their effect on a2, 6 sialylation at the cell surface. A. Expression plasmids for full-length WT ST6Gal I (WT), double-mutant ST6Gal I (L37A/K40A) and N-terminal ST6Gal I deletion mutant (ΔN, containing amino acids 43-403) were constructed. WT and L37A/K40A were N-terminally tagged with Flag; ΔN was tagged with HA. Expression of each construct was confirmed by immunoblotting. SW480 cells were transfected with WT, L37A/K40A, or ΔN. Then, ST6Gal I enzymatic activity was measured by ELISA (B), and cell surface sialylation was detected by FACS (C). Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). to a greater degree than did the WT form. Next, we of the cell surface glycoprotein integrin b1, it signifi- further examined the effects of BACE1 on ST6Gal I cantly induced p130CAS phosphorylation and migration cleavage by co-transfecting SW480 cells with BACE1 of SW480 cells (Figure 4A and 4B) and CT26 cells (data and WT or L37A/K40A forms of ST6Gal I. These not shown), similar to the results of our previous study experiments showed that the Golgi-anchored WT pro- [23,26]. These findings suggest that the soluble form of form was decreased to a greater extent by co-transfec- ST6Gal I could be involved in p130CAS signaling and tion of BACE1 compared with the non-cleavable L37A/ migration of colon cancer cells, which, in turn, may be K40A form (Figure 3B). We then extended our investi- independent of integrin b1 sialylation. gation of the roles of BACE1 in ST6Gal I-mediated migration of cancer cells by co-transfecting SW480 and BACE-1 mediated cleavage and secretion of ST6Gal I are CT26 cells with BACE1 and WT or L37A/K40A. Cell increased by IR migration was increased to a much lesser extent by co- Our previous study indicated that IR induces ST6Gal I expression of BACE1 and WT compared with coexpres- cleavage and secretion of a soluble form of ST6Gal I sion of BACE1 and L37A/K40A (Figure 3C). These find- [23]. Because it has been suggested that BACE1 is a pro- ings demonstrate that BACE-1 is capable of cleaving the tease of ST6Gal I, we tested the effect of IR on BACE1 Golgi-membrane anchored ST6Gal I, and indicate that expression. Importantly, IR (10 Gy) increased the BACE-1-induced cleavage of ST6Gal I results in expression of BACE1 at both mRNA and protein levels decreased cell migration. in SW480 and CT26 colon cancer cell lines (Figure 5A). Next, we examined whether IR induced ST6Gal I clea- The soluble form of ST6Gal I is partially involved in the vage andproducedasolubleformofST6GalI. As migration of colon cancer cells shown in Figure 5B, IR increased cleavage of ST6Gal I Next, we compared the effects of ST6Gal I WT and ΔN and knockdown of BACE1 inhibited this cleavage. In on integrin b1 sialylation, integrin b1-mediated signal- both SW480 and CT26 cell lines, the enzymatic activity ing, and cell migration. Expression of either WT or ΔN of ST6Gal I in culture media was increased compared to increased the levels of the Tyr410-phosphorylated that in control cells after exposure of vector control or Y410 (active) form of p130CAS (p-p130CAS ;Figure4A) ST6Gal I-overexpressing cells to IR (Figure 5C). How- and enhanced cell migration (Figure 4B). Although the ever, even though IR alone slightly increased cell surface ΔN soluble form had no significant effects on sialylation protein sialylation which might be induction of ST6Gal Lee et al. Radiation Oncology 2012, 7:47 Page 6 of 10 http://www.ro-journal.com/content/7/1/47 Figure 3 Sialylation and migration effects of non-cleavable ST6Gal I (L37A/K40A). A. SW480 cells were transfected with WT ST6Gal I (WT) Y410 or ST6Gal I (L37A/K40A) and cell lysates were analyzed for the phosphorylated form of p130CAS (p-P130CAS ) and integrin b1by immunoblotting. Expression of ST6Gal I was assessed using an anti-Flag antibody. Sialylation of integrin b1 was determined by SNA lectin affinity assay. B. SW480 cells were co-transfected with BACE1 and the WT or L37A/K40A form of ST6Gal I. Cell lysates were immunoblotted with antibodies against BACE1 and Flag. C. SW480 cells and CT26 cells were co-transfected with BACE1 and WT or L37A/K40A. Twenty-four hours after transfection, cell migration was determined using Transwell migration assays. Upper: images of migrated cells captured by phase-contrast microscopy; lower: migration measured by counting cells. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). I by IR [23,24], Golg-anchored ST6Gal I-mediated sialylation and migration were partially inhibited by IR, increase of cell surface sialylation was not augmented by an effect that might be due to cleavage of ST6Gal I (Fig- IR (Figure 5D). We also checked the effect of IR on ure 5E). ST6Gal I-induced cell migration. ST6Gal I enhanced CT26 cell migration, an effect that was partially inhib- Discussion ited by IR. These results indicate that IR acted through In the present study, we showed that IR induced clea- induction of BACE1 expression to induce cleavage of vage and secretion of the soluble form of ST6Gal I by ST6Gal I protein to a soluble form that retained sialyl- increasing BACE-1 expression, an effect that may contri- transferase activity. ST6Gal I-induced cell surface bute to IR-induced migration of colon cancer cells, Lee et al. Radiation Oncology 2012, 7:47 Page 7 of 10 http://www.ro-journal.com/content/7/1/47 Figure 4 The soluble form of ST6Gal I increases the migration of colon cancer cells. A. SW480 cells were transfected with WT ST6Gal I Y410 (WT) or ST6Gal I-ΔN(ΔN), and cell lysates were analyzed for the phosphorylated form of p130CAS (p-P130CAS ) and integrin b1by immunoblotting. Sialylation of integrin b1 was determined by SNA lectin affinity assay. B. After transfection with the WT or ΔN form of ST6Gal I, cell migration was determined using Transwell migration assays. Upper: images of migrated cells captured by phase-contrast microscopy; lower: migration measured by counting cells. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). which phenomena is independent of integrin b1sialyla- tissues. Information regarding BACE-1 expression and tion that was usually mediated by Golgi-anchored functions outside of the brain is limited. Furthermore, ST6Gal I. the physiological importance of BACE-1 in cancer pro- Our previous study suggested that IR increases the gression, metastasis, and responsiveness to radiation expression of ST6Gal I, which, in turn, is involved in remains largely unknown. Evidence that BACE-1 is one radioresistance and radiation-induced migration via sia- of the proteases responsible for the cleavage of ST6Gal I lylation of integrin b1 [22,23,26]. Another interesting was provided by experiments employing a b-secretase issue raised previously is that IR increases ST6Gal I inhibitororsiRNA-mediatedknockdown of BACE-1 cleavage and secretion into culture media [23]. While (Figure 1). The results of these experiments suggest that the current data indicate that the Golgi is the main site BACE1 activity involved in cleavage of ST6Gal I from of sialylation by ST6Gal I in most cells, the fact that the Golgi membrane and inducing secretion. soluble forms of ST6Gal I are detected in body fluids Interestingly, inhibition of g-secretase also increased and media from cultured cells has raised questions con- ST6Gal I levels and cell surface sialylation. Although cerning the functions of this extracellular ST6Gal I. It is BACE-1 expression levels could be a major determinant known that ST6Gal I is not the only transferase that of ST6Gal I cleavage, other regulatory mechanisms exists in a soluble form; other glycosyltransferases are might also affect cleavage, secretion, or sorting of also detected in the systemic circulation of cancer ST6Gal I to critical subcellular localizations where patients, where they are associated with disease severity ST6Gal I could encounter proteases. Such BACE-1- and poor prognosis. It has also been suggested that independent mechanisms might also regulate ST6Gal I soluble ST6Gal I may be a potential biomarker for the function. clinical evaluation of colorectal cancer, highlighting the In colon cancer cells, IR increased the expression of importance of elucidating the function of soluble BACE1, which is involved in IR-mediated cleavage of ST6Gal I, particularly in the radiation-induced migration ST6Gal I (Figure 5). To address the detailed mechan- of cancer cells. isms linking ST6Gal I to cancer cell migration, we BACE-1, which is a crucial protease in the pathogen- established non-cleavable (L37A/K40A) and soluble esis of Alzheimer’sdisease,ishighlyexpressed in the (ΔN) variants of ST6Gal I that retained catalytic activity brain, but it is also expressed at low levels in peripheral (Figure 2). Cleaved ST6Gal I was secreted into the Lee et al. Radiation Oncology 2012, 7:47 Page 8 of 10 http://www.ro-journal.com/content/7/1/47 Figure 5 IR-induced BACE-1 mediates cleavage of ST6Gal I, and inhibits sialylation of cell surface molecules and colon cancer cell migration. A. Following irradiation of SW480 and CT26 cells, BACE1 expression levels were determined by RT-PCR and immunoblotting. CT26 vector control cells and cells stably expressing ST6Gal I were exposed to 10 Gy of IR. B. The soluble form of ST6Gal I was analyzed after co- transfection with WT ST6Gal I and BACE1, or after exposure of IR (10 Gy, 48 h) to WT ST6Gal I -transfected cells. C. SW480 and CT26 colorectal carcinoma cell lines were stably transfected with ST6Gal I and exposed to 10 Gy of IR. After 24 hours, culture media were harvested and ST6Gal I activity was assayed by ELISA. Data are presented as means ± SDs from three replicates. CT26 vector control cells and cells stably expressing ST6Gal I were exposed to 10 Gy of IR. After 24, a2,6 sialylation of cell surface was analyzed by flow cytometry. Data are presented as means ± SDs of three replicates. *p < 0.05. n.s., not significant. (D). Cell migration was determined using Transwell migration assays (E). Upper: images of migrated cells captured by phase-contrast microscopy; lower: migration measured by counting cells. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). Lee et al. Radiation Oncology 2012, 7:47 Page 9 of 10 http://www.ro-journal.com/content/7/1/47 Authors’ contributions culture media after exposure to radiation (Figure 5), and ML and JJP performed experiments. YGK and YSL designed, analyzed data, the truncated soluble ΔN form retained sialyltransferase and wrote paper. All authors have read and approved the final manuscript. activity (Figure 2). We had predicted that the soluble Competing interests form of ST6Gal I was merely a byproduct of the Golgi- The authors declare that they have no competing interests. anchored proform of ST6Gal I that was excreted into the extracellular milieu after exposure of cells to radia- Received: 1 December 2011 Accepted: 27 March 2012 Published: 27 March 2012 tion. However, ST6Gal I-ΔN also induced cellular migration (Figure 4), suggesting that the soluble form of References ST6Gal I could function in tumor cell migration with 1. 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Stewart JF, Rubens RD, Hoare S, Bulbrook RD, Kessel D: Serum sialyl Conclusion transferase levels in patients with metastatic breast cancer treated by Collectively, our data suggest that glycoconjugation by chemotherapy. British journal of cancer 1982, 46:208-212. 10. Bernacki RJ, Kim U: Concomitant elevations in serum sialytransferase the Golgi form of ST6Gal I, possibly in cooperation activity and sialic acid content in rats with metastasizing mammary with soluble ST6Gal I, may be involved in the process tumors. Science (New York, N.Y 1977, 195:577-580. of cancer metastasis, especially after radiation therapy. 11. Gessner P, Riedl S, Quentmaier A, Kemmner W: Enhanced activity of CMP- neuAc: Gal beta 1-4GlcNAc:alpha 2,6-sialyltransferase in metastasizing human colorectal tumor tissue and serum of tumor patients. Cancer Funding letters 1993, 75:143-149. This work was supported by the Nuclear Research and 12. Ganzinger U, Moser K: Sialyl transferase activity: a serum enzyme marker in the follow-up of cancer patients. Recent results in cancer research. Development Program through a National Research Fortschritte der Krebsforschung 1979, 67:50-55. Foundation of Korea (NRF) grant funded by the Korean 13. Herrmann WP, Gielen W: Sialyltransferase levels and sialic acid government (Ministry of Education, Science and Tech- concentrations in sera of patients with malignant melanomas. Archives of dermatological research 1979, 265:321-329. nology; grant code: M2AMA006), and by a grant from 14. Kawaguchi T: Cancer metastasis: characterization and identification of the Korea Healthcare Technology R&D Project, Ministry the behavior of metastatic tumor cells and the cell adhesion molecules, for Health, Welfare & Family Affairs (grant code: including carbohydrates. Curr Drug Targets Cardiovasc Haematol Disord 2005, 5:39-64. A100627). This work was also supported by the Ewha 15. Hakomori S: Tumor malignancy defined by aberrant glycosylation and Global Top5 Grant 2011 of Ewha Womans University. sphingo(glyco)lipid metabolism. Cancer research 1996, 56:5309-5318. 16. Zhao YY, Takahashi M, Gu JG, Miyoshi E, Matsumoto A, Kitazume S, Taniguchi N: Functional roles of N-glycans in cell signaling and cell Author details adhesion in cancer. Cancer science 2008, 99:1304-1310. Division of Radiation Effects, Korea Institute of Radiological and Medical 17. Gorelik E, Galili U, Raz A: On the role of cell surface carbohydrates and Sciences, Seoul 139-706, South Korea. College of Life Sciences and their binding proteins (lectins) in tumor metastasis. Cancer Metastasis Rev Biotechnology, Korea University, 1, 5-ka, Anamdong, Sungbuk-gu, Seoul 136- 2001, 20:245-277. 701, South Korea. College of Pharmacy & Division of Life & Pharmaceutical 18. Semel AC, Seales EC, Singhal A, Eklund EA, Colley KJ, Bellis SL: Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu, Hyposialylation of integrins stimulates the activity of myeloid fibronectin Seoul 120-750, South Korea. receptors. J Biol Chem 2002, 277:32830-32836. Lee et al. Radiation Oncology 2012, 7:47 Page 10 of 10 http://www.ro-journal.com/content/7/1/47 19. Seales EC, Jurado GA, Singhal A, Bellis SL: Ras oncogene directs expression 41. Cordes N, Seidler J, Durzok R, Geinitz H, Brakebusch C: beta1-integrin- of a differentially sialylated, functionally altered beta1 integrin. Oncogene mediated signaling essentially contributes to cell survival after radiation- 2003, 22:7137-7145. induced genotoxic injury. Oncogene 2006, 25:1378-1390. 20. van der Flier A, Sonnenberg A: Function and interactions of integrins. Cell doi:10.1186/1748-717X-7-47 Tissue Res 2001, 305:285-298. Cite this article as: Lee et al.: Cleavage of ST6Gal I by Radiation-Induced 21. Van Slambrouck S, Grijelmo C, De Wever O, Bruyneel E, Emami S, BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin Gespach C, Steelant WF: Activation of the FAK-src molecular scaffolds b1 and Migration in Colon Cancer Cells. Radiation Oncology 2012 7:47. and p130Cas-JNK signaling cascades by alpha1-integrins during colon cancer cell invasion. Int J Oncol 2007, 31:1501-1508. 22. Lee M, Park JJ, Lee YS: Adhesion of ST6Gal I-mediated human colon cancer cells to fibronectin contributes to cell survival by integrin beta1- mediated paxillin and AKT activation. Oncol Rep 2010, 23:757-761. 23. Lee M, Lee HJ, Bae S, Lee YS: Protein sialylation by sialyltransferase involves radiation resistance. Mol Cancer Res 2008, 6:1316-1325. 24. Lee M, Lee HJ, Seo WD, Park KH, Lee YS: Sialylation of integrin beta1 is involved in radiation-induced adhesion and migration in human colon cancer cells. Int J Radiat Oncol Biol Phys 2010, 76:1528-1536. 25. Jang ER, Ryu M, Park JE, Kim JH, Lee JS, Song K: A new isoquinolinium derivative, Cadein1, preferentially induces apoptosis in p53-defective cancer cells with functional mismatch repair via a p38-dependent pathway. J Biol Chem 2010, 285:2986-2995. 26. Lee M, Lee HJ, Seo WD, Park KH, Lee YS: Sialylation of integrin beta1 is involved in radiation-induced adhesion and migration in human colon cancer cells. Int J Radiat Oncol Biol Phys 2010, 76:1528-1536. 27. Vassar R, Kovacs DM, Yan R, Wong PC: The beta-secretase enzyme BACE in health and Alzheimer’s disease: regulation, cell biology, function, and therapeutic potential. J Neurosci 2009, 29:12787-12794. 28. Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, et al: Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science (New York, N.Y 1999, 286:735-741. 29. Kitazume S, Oka R, Ogawa K, Futakawa S, Hagiwara Y, Takikawa H, Kato M, Kasahara A, Miyoshi E, Taniguchi N, Hashimoto Y: Molecular insights into beta-galactoside alpha2,6-sialyltransferase secretion in vivo. Glycobiology 2009, 19:479-487. 30. Strous GJ: Golgi and secreted galactosyltransferase. CRC critical reviews in biochemistry 1986, 21:119-151. 31. Paone JF, Waalkes TP, Baker RR, Shaper JH: Serum UDP-galactosyl transferase as a potential biomarker for breast carcinoma. Journal of surgical oncology 1980, 15:59-66. 32. De AK, Hardy RE: Elucidation of sialyltransferase as a tumour marker. Indian journal of biochemistry & biophysics 1990, 27:452-455. 33. Futakawa S, Kitazume S, Oka R, Ogawa K, Hagiwara Y, Kinoshita A, Miyashita K, Hashimoto Y: Development of sandwich enzyme-linked immunosorbent assay systems for plasma beta-galactoside alpha2,6- sialyltransferase, a possible hepatic disease biomarker. Analytica chimica acta 2009, 631:116-120. 34. Kitazume S, Suzuki M, Saido TC, Hashimoto Y: Involvement of proteases in glycosyltransferase secretion: Alzheimer’s beta-secretase-dependent cleavage and a following processing by an aminopeptidase. Glycoconjugate journal 2004, 21:25-29. 35. Sugimoto I, Futakawa S, Oka R, Ogawa K, Marth JD, Miyoshi E, Taniguchi N, Hashimoto Y, Kitazume S: Beta-galactoside alpha2,6-sialyltransferase I cleavage by BACE1 enhances the sialylation of soluble glycoproteins. A novel regulatory mechanism for alpha2,6-sialylation. The Journal of biological chemistry 2007, 282:34896-34903. 36. Lee HW, Seo HS, Ha I, Chung SH: Overexpression of BACE1 stimulates spontaneous basal secretion in PC12 cells. Neuroscience letters 2007, 421:178-183. Submit your next manuscript to BioMed Central 37. Pietras RJ, Szego CM, Mangan CE, Seeler BJ, Burtnett MM, Orevi M: Elevated and take full advantage of: serum cathepsin B1 and vaginal pathology after prenatal DES exposure. Obstetrics and gynecology 1978, 52:321-327. • Convenient online submission 38. Seales EC, Shaikh FM, Woodard-Grice AV, Aggarwal P, McBrayer AC, Hennessy KM, Bellis SL: A protein kinase C/Ras/ERK signaling pathway • Thorough peer review activates myeloid fibronectin receptors by altering beta1 integrin • No space constraints or color figure charges sialylation. J Biol Chem 2005, 280:37610-37615. • Immediate publication on acceptance 39. Cordes N, Blaese MA, Meineke V, Van Beuningen D: Ionizing radiation induces up-regulation of functional beta1-integrin in human lung • Inclusion in PubMed, CAS, Scopus and Google Scholar tumour cell lines in vitro. Int J Radiat Biol 2002, 78:347-357. • Research which is freely available for redistribution 40. Cordes N, Park CC: beta1 integrin as a molecular therapeutic target. Int J Radiat Biol 2007, 83:753-760. 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Cleavage of ST6Gal I by Radiation-Induced BACE1 Inhibits Golgi-Anchored ST6Gal I-Mediated Sialylation of Integrin β1 and Migration in Colon Cancer Cells

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Springer Journals
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Copyright © 2012 by Lee et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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1748-717X
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10.1186/1748-717X-7-47
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22449099
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Abstract

Background: Previously, we found that b-galactoside a2,6-sialyltransferase (ST6Gal I), an enzyme that adds sialic acids to N-linked oligosaccharides of glycoproteins and is frequently overexpressed in cancer cells, is up-regulated by ionizing radiation (IR) and cleaved to a form possessing catalytic activity comparable to that of the Golgi- localized enzyme. Moreover, this soluble form is secreted into the culture media. Induction of ST6Gal I significantly increased the migration of colon cancer cells via sialylation of integrin b1. Here, we further investigated the mechanisms underlying ST6Gal I cleavage, solubilization and release from cells, and addressed its functions, focusing primarily on cancer cell migration. Methods: We performed immunoblotting and lectin affinity assay to analyze the expression of ST6 Gal I and level of sialylated integrin b1. After ionizing radiation, migration of cells was measured by in vitro migration assay. a2, 6 sialylation level of cell surface was analyzed by flow cytometry. Cell culture media were concentrated and then analyzed for soluble ST6Gal I levels using an a2, 6 sialyltransferase sandwich ELISA. Result: We found that ST6Gal I was cleaved by BACE1 (b-site amyloid precursor protein-cleaving enzyme), which was specifically overexpressed in response to IR. The soluble form of ST6Gal I, which also has sialyltransferase enzymatic activity, was cleaved from the Golgi membrane and then released into the culture media. Both non- cleaved and cleaved forms of ST6Gal I significantly increased colon cancer cell migration in a sialylation-dependent manner. The pro-migratory effect of the non-cleaved form of ST6Gal I was dependent on integrin b1 sialylation, whereas that of the cleaved form of ST6Gal I was not, suggesting that other intracellular sialylated molecules apart from cell surface molecules such as integrin b1 might be involved in mediating the pro-migratory effects of the soluble form of ST6Gal I. Moreover, production of soluble form ST6Gal I by BACE 1 inhibited integrin b1 sialylation and migration by Golgi-anchored form of ST6Gal I. Conclusions: Our results suggest that soluble ST6Gal I, possibly in cooperation with the Golgi-bound form, may participate in cancer progression and metastasis prior to being secreted from cancer cells. Keywords: BACE1, Migration, Radiation, ST6Gal I * Correspondence: ygko@korea.ac.kr; yslee0425@ewha.ac.kr † Contributed equally College of Life Sciences and Biotechnology, Korea University, 1, 5-ka, Anamdong, Sungbuk-gu, Seoul 136-701, South Korea College of Pharmacy & Division of Life & Pharmaceutical Sciences, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemun-Gu, Seoul 120-750, South Korea Full list of author information is available at the end of the article © 2012 Lee et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lee et al. Radiation Oncology 2012, 7:47 Page 2 of 10 http://www.ro-journal.com/content/7/1/47 forms of glycosyltransferases exist in the plasma of Background patients with certain diseases, and can sometimes be ST6Gal I (b galactoside a2,6 sialyltransferase, CMP- used as biomarkers for these diseases [30-33]. NeuAc: Galb (1,4) GlcNAc: a2,6 sialyltransferase) is an In the present study, we examined IR-induced clea- important glycosyltransferase that adds a sialic acid resi- vage and solubilization of ST6Gal I, which is released due to the terminal position on N-linked oligosacchar- into the cell culture media of colon cancer cell lines, ides [1,2]. It is localized in the Golgi apparatus in a and sought to identify the protease involved in cleaving membrane-anchored form and is cleaved into a secre- ST6Gal I after exposure to IR. We found that BACE1 tary protein by cathepsin-like proteases [3]. Recent stu- could be the secretase responsible for IR-induced clea- dies and clinical reports have emphasized the vage of ST6Gal I, and showed that BACE1 mediated importance of ST6Gal I in cancer progression and cleavage of ST6Gal I decreased ST6Gal I -mediated can- metastasis. ST6Gal I is up-regulated in colon adenocar- cer cell migration. Additionally, the soluble form of cinoma and its expression is positively associated with ST6Gal I possesses sialyltransferase enzymatic activity, tumor cell migration and invasion [4-6]. Specifically, but unlike the Golgi-associated form, did not sialylate patients with metastasizing tumors have high levels of integrin b1 to a significant degree, suggesting that solu- ST6Gal I in their serum, and serum levels of ST6Gal I ble ST6Gal I, possibly in cooperation with the Golgi- are correlated with the progression of colorectal carci- bound form, may participate in cancer progression and nomas and cancer metastasis [7-13]. However, a possible metastasis before being secreted from cancer cells, inde- biological role of ST6Gal I in the plasma has not been pendent of intergrin b1-sialylation. reported. Metastasis represents an obligatory step in cancer Materials and methods progression. A variety of molecules contribute to can- Cell culture cer progression and metastasis [14], and many of the SW480 and SW48 human colorectal carcinoma cells factors that function in tumor metastasis are glycopro- were growninRPMImedia supplemented with heat- teins [15-17]. It has been previously demonstrated that inactivated 10% fetal bovine serum and antibiotics. integrin b1 is a major substrate of ST6Gal I [4,18]. In CT26 mouse colorectal carcinoma cells were cultured in colon epithelial cells, oncogenic Ras has been shown to Dulbecco’s modified Eagle’s medium (DMEM) supple- up-regulate ST6Gal I expression, leading to increased mented with heat-inactivated 10% fetal bovine serum a-2,6 sialylation of b1 integrin [19]. Hypersialylation of and antibiotics. integrin b1 augments colon cancer metastasis by alter- ing cellular preference for a certain extracellular matrix milieu as well as by stimulating cell migration. Integ- Plasmids and transfection For transient transfection experiments, expression con- rins also regulate cellular functions, including survival, structs of C-terminally Flag-tagged wild-type (WT; p3 × proliferation and cell spreading, through the function Flag-ST6Gal I) ST6Gal I and N-terminally deleted (HA- of signaling molecules co-localized to the focal adhe- tagged) ST6Gal I (ST6Gal I ΔN) were created as sion complex [20,21]. We have previously demon- described previously [23]. An N-terminally Flag-tagged strated that exposure to ionizing radiation (IR) double-mutant, ST6Gal I (L37A/K40A) was created by increases the expression of ST6Gal I as well as the site-directed mutagenesis [34]. Predesigned small inter- level of sialylated glycoprotein. Sialylation of integrin fering RNA (siRNA) for ST6Gal I was purchased from b1 by exposure of cells to IR increases the adhesion Dharmacon (Lafayette, CO, USA). BACE-1 was targeted and migration of colon cancer cells through integrin with the siRNA duplex 5’-AGA UCC UGU CCA UUG b1-mediated cellular signaling. Therefore, integrin b1 AU CUC CAC CC-3 ’ and 5 ’-GGG UGG AGA UCA sialylation and the subsequent activation of p130CAS, AUG GAC AGG AUC U-3’ [35]. A BACE-1 expression paxillin, and AKT signaling may be one of the plasmid (pcDNA3.1/BACE-1) was generously provided mechanisms involved in IR-mediated-radioresistance by Dr. Sul-Hee Chung (Inje University, Pusan, Korea) and cancer metastasis [22-26]. [36]. Cells were transfected with plasmids using Lipofec- b-site amyloid precursor protein-cleaving enzyme tAMINE 2000 (Invitrogen, Carlsbad, CA, USA) as (BACE) is a membrane-bound aspartic protease that described by the manufacturer. cleaves the amyloid precursor protein (APP) in the pathogenesis of Alzheimer’s disease [27,28]. Importantly, Irradiation BACE has been identified as a protease responsible for Cells were exposed to g-irradiation using a Cs g-ray the cleavage and secretion of Golgi-resident ST6Gal I [29]. The mechanisms underlying cleavage are compli- source (Atomic Energy of Canada, Mississauga, ON, cated, and have not been well characterized. Soluble Canada) at a dose rate of 3.81 Gy/min. Lee et al. Radiation Oncology 2012, 7:47 Page 3 of 10 http://www.ro-journal.com/content/7/1/47 Immunoblot, lectin affinity assay, and and then analyzed for soluble ST6Gal I levels using an immunoprecipitation a2,6 sialyltransferase sandwich enzyme-linked immuno- Protein levels were detected using the following commer- sorbent assay (ELISA) kit (IBL, Japan), according to the cial antibodies: anti-integrin b1 (BD Biosciences, Franklin manufacturer’s protocol. Lakes, NJ, USA); anti-phospho-p130CAS, anti-phospho- Src, anti-HA, and anti-Myc (Cell Signaling Technology, Statistical analysis Danvers, MA, USA); anti-Flag (Sigma); and anti-ST6Gal I Data are expressed as means ± standard deviations and anti-BACE-1 (IBL, Japan). For detection of sialylated (SDs). Statistical significance was determined using Stu- proteins, cell lysates were incubated with biotinylated SNA dent’s t-test for comparisons between two means. The (Sambucus Nigra lectin; Vector Laboratories, Burlingame, null hypothesis was rejected in cases where p-values CA, USA), and protein-lectin complexes were precipitated were < 0.05. with avidin-coated protein A-agarose (Sigma). Results Flow cytometry Inhibition of ST6Gal I cleavage by knockdown of BACE1 Cells were detached with trypsin/EDTA at the indicated increases sialylation of cell surface protein times and stained with fluorescein isothiocyanate In our previous study, we found that IR exposure (FITC)-conjugated SNA (FITC-SNA) or Maackia amur- induces ST6Gal I cleavage and produces a soluble form ensis (FITC-MAA) lectin (Vector Laboratories, Burlin- of ST6Gal I in SW480 colon cancer cells [23]. To sup- game, CA, USA) for detection of a2,6 and a2,3 port the result of our prior work, we next examined the sialylation, respectively. After staining, fluorescence expression of soluble ST6 Gal I in SW48 cells (ST6 Gal -/- intensity was analyzed by fluorescence-activated cell I ) by transiently transfecting these cells with expres- sorting (FACS). sion vectors for Golgi-anchored (N-terminally Flag- tagged and C-terminally Myc-tagged) of ST6Gal I and Reverse transcription-polymerase chain reaction (RT-PCR) exposing them to IR. As shown in Figure 1A, both the Total RNA was isolated with TRI reagents (Molecular Golgi-anchored and soluble forms of ST6Gal I were Research Center), and reverse transcription was done expressed in SW48 colon cancer cells (Figure 1A). using Omniscript transcriptase (Qiagen) under the fol- Because it had previously been demonstrated that sialyl- lowing thermocycling conditions: 30 cycles of 95°C for 5 transferases are cleaved by cathepsin-like protease or min (denaturing), 60°C for 30 s (annealing), and 72°C BACE [3,29,34,37], we sought to identify the target pro- for 30 seconds (extension). The primer sequences used tease(s) responsible for cleavage of ST6Gal I using var- were as follows: human BACE1, 5’-GGT GGA GAT ious protease inhibitors in SW480 vector-control and CAA TGG ACA GG-3’ (sense) and 5’-CGT GGA TGA ST6Gal I-overexpressing cells. Inhibitors of b-secretase CTG TGA GAT GG-3’ (antisense); mouse Bace1, 5’- and g-secretase increased the protein levels of the GCAGAC CCACAT TCCGAA CA-3’ (sense) and 5’- uncleaved, proform of ST6Gal I, whereas cathepsin B GCC ACT GTC CAC GAT GCT CTT-3’ (anti-sense); and L (CTS) inhibitors did not (Figure 1B). In addition, and GAPDH, 5’-CAT GGA GAA GGC TGG GGC TCA a2, 6 sialylation level at the cell surface, detected by TTT-3’ (sense) and 5 ’-CGC CAG TAG AGG CAG FITC-conjugated SNA, was also increased by treatment GGA TGA TGT-3’ (antisense). with b-secretase or g-secretase, but not CTS, inhibitors. In contrast, a2, 3 sialylation at the cell surface, detected In vitro migration assay by FITC-MAA, was not altered by these inhibitors (Fig- Cell migration assays were performed using a Boyden ure 1C), suggesting that b-secretase or g-secretase might chamber as previously described [24]. Cells were plated be responsible for the cleavage of ST6Gal I in SW480 on the upper side of a collagen treated, polycarbonate colon cancer cells. Next, because BACE1 is reported to membrane separating two chambers of 6.5-mm Trans- be the protease responsible for the cleavage of ST6Gal 1 well culture plates (Costar, Corning, NY, USA). After 24 [34,35], we tested the effects of siRNA-mediated BACE1 hours, cells on the upper face of the membrane were knockdown. We observed a concomitant increase in the scraped using a cotton swab and cells that had migrated level of non-cleaved ST6Gal I protein and sialylation of to the lower face of the membrane were stained with integrin b1, a substrate of ST6Gal I (Figure 1D). More- DiffQuick (Baxter Scientific, Deerfield, IL, USA) Wright- over, knockdown of BACE1 expression augmented the Giemsa Solution. sialylationonthesurfaceofthese cells. Interestingly, BACE1 overexpression did not specifically inhibit cell Soluble ST6Gal I ELISA surface sialylation (and may have slightly increased it) Cell culture media were concentrated using Centricon compared with control levels (Figure 1E), suggesting centrifugal filter devices (Millipore, Billerica, MA, USA) that BACE1-mediated cleavage of ST6Gal I only slightly Lee et al. Radiation Oncology 2012, 7:47 Page 4 of 10 http://www.ro-journal.com/content/7/1/47 Figure 1 Inhibition of BACE1 stabilizes ST6Gal I and increases cell surface sialylation. A. SW48 (ST6Gal I-negative) colon cancer cells were transiently transfected with an N-terminally Flag-tagged ST6Gal I expression plasmid, and 24 hours later, cells were exposed to IR (10 Gy). Cell lysates were assessed for expression of ST6Gal I by immunoblotting with an ST6Gal I antibody. B. SW480 vector-control cells and cells stably expressing ST6Gal I were treated with inhibitors of b-secretase, g-secretase, or cathepsin B&L at a concentration of 20 μM. After incubating for 24 hours, cells were harvested and ST6Gal I levels were analyzed by immunoblotting with an anti-ST6Gal I antibody. C. Cell surface a2, 6 sialylation (SNA-FITC) and a2, 3 sialylation (MAA-FITC) were analyzed by FACS after 24 hours of treatment with 20 μM b-secretase or g-secretase inhibitors. D. SW480 cells were transfected with siRNA against BACE1, and the levels of BACE1, ST6Gal I, and integrin b1 protein were assessed by immunoblotting. Sialylation of integrin b1 was assayed by SNA lectin affinity assay. E. SW480 vector-control cells and cells stably expressing ST6Gal I were treated with Si-BACE1 or transfected with a BACE1 expression plasmid. Then, a2, 6 sialylation (SNA-FITC) of the cell surface was analyzed by FACS. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). shows the sialylation potential of ST6Gal I. Taken glycoproteins were barely sialylated compared to WT or together, these results suggest that BACE1 may be L37A/K40A (Figure 2C). These results show that the responsible for cleavage of ST6Gal I. non-cleavable form of ST6Gal I has enzymatic activity and increases a2, 6 sialylation at the cell surface to the The non-cleavable, double-mutant ST6Gal I (L37A/K40A) same extent as WT. However, it was suggested that the increases a2, 6 sialylation at the cell surface sialylation targets of the soluble form of ST6Gal I were It was previously demonstrated that replacement of not cell surface proteins. This result may be due to the ST6Gal I residues Leu37 and Lys40 with alanine signifi- fact that there isn’t much CMP-sialic acid substrate on cantly decreases BACE1-mediated cleavage of ST6Gal I the outside of the cell. [34]. Therefore, we prepared a N-terminally Flag-tagged and C-terminally myc-tagged, non-cleavable ST6Gal I Non-cleaved form ST6Gal I shows integrin b1 dependent (L37A/K40A)mutantandan HA-tagged, N-terminally cell migration truncated soluble form of ST6Gal I (ΔN, amino acids Integrin b1, aknownsubstrateofST6GalI[4,38], 43-403) lacking the protease-specific recognition motif together with associated adaptor molecules, such as (Figure 2A). We then transiently transfected cells with p130CAS, plays a role in promoting the migration of ST6Gal I WT, L37A/K40A or ΔN constructs, and exam- cancer cells [39-41]. We previously reported that integ- ined ST6Gal I protein expression by immunoblotting rin b1 sialylation and its associated cancer cell migra- (Figure 2A) and measured ST6Gal I enzymatic activity tion-related signaling activity are increased by IR- and sialylation of cell surface proteins 24 hours later. induced ST6Gal I [22,23,26]. The phenomena described We found that the enzymatic activity of both the non- above prompted us to compare the action of WT and cleavable L37A/K40A mutant and the ΔNsoluble form non-cleavable L37A/K40A on integrin b1 sialylation, was similar to that of WT (Figure 2B). In addition, integrin b1-mediated signaling, and cell migration. As L37A/K40A increased cell surface sialylation to a similar shown in Figure 3A, transfection with L37A/K40A extent as WT. However, in the case of ΔN, which also increased the Tyr410-phosphorylated (active) form of Y410 had enzymatic catalytic activity, cell surface p130CAS (p-p130CAS ) and sialylation of integrin b1 Lee et al. Radiation Oncology 2012, 7:47 Page 5 of 10 http://www.ro-journal.com/content/7/1/47 Figure 2 Construction of ST6Gal I expression plasmids and their effect on a2, 6 sialylation at the cell surface. A. Expression plasmids for full-length WT ST6Gal I (WT), double-mutant ST6Gal I (L37A/K40A) and N-terminal ST6Gal I deletion mutant (ΔN, containing amino acids 43-403) were constructed. WT and L37A/K40A were N-terminally tagged with Flag; ΔN was tagged with HA. Expression of each construct was confirmed by immunoblotting. SW480 cells were transfected with WT, L37A/K40A, or ΔN. Then, ST6Gal I enzymatic activity was measured by ELISA (B), and cell surface sialylation was detected by FACS (C). Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). to a greater degree than did the WT form. Next, we of the cell surface glycoprotein integrin b1, it signifi- further examined the effects of BACE1 on ST6Gal I cantly induced p130CAS phosphorylation and migration cleavage by co-transfecting SW480 cells with BACE1 of SW480 cells (Figure 4A and 4B) and CT26 cells (data and WT or L37A/K40A forms of ST6Gal I. These not shown), similar to the results of our previous study experiments showed that the Golgi-anchored WT pro- [23,26]. These findings suggest that the soluble form of form was decreased to a greater extent by co-transfec- ST6Gal I could be involved in p130CAS signaling and tion of BACE1 compared with the non-cleavable L37A/ migration of colon cancer cells, which, in turn, may be K40A form (Figure 3B). We then extended our investi- independent of integrin b1 sialylation. gation of the roles of BACE1 in ST6Gal I-mediated migration of cancer cells by co-transfecting SW480 and BACE-1 mediated cleavage and secretion of ST6Gal I are CT26 cells with BACE1 and WT or L37A/K40A. Cell increased by IR migration was increased to a much lesser extent by co- Our previous study indicated that IR induces ST6Gal I expression of BACE1 and WT compared with coexpres- cleavage and secretion of a soluble form of ST6Gal I sion of BACE1 and L37A/K40A (Figure 3C). These find- [23]. Because it has been suggested that BACE1 is a pro- ings demonstrate that BACE-1 is capable of cleaving the tease of ST6Gal I, we tested the effect of IR on BACE1 Golgi-membrane anchored ST6Gal I, and indicate that expression. Importantly, IR (10 Gy) increased the BACE-1-induced cleavage of ST6Gal I results in expression of BACE1 at both mRNA and protein levels decreased cell migration. in SW480 and CT26 colon cancer cell lines (Figure 5A). Next, we examined whether IR induced ST6Gal I clea- The soluble form of ST6Gal I is partially involved in the vage andproducedasolubleformofST6GalI. As migration of colon cancer cells shown in Figure 5B, IR increased cleavage of ST6Gal I Next, we compared the effects of ST6Gal I WT and ΔN and knockdown of BACE1 inhibited this cleavage. In on integrin b1 sialylation, integrin b1-mediated signal- both SW480 and CT26 cell lines, the enzymatic activity ing, and cell migration. Expression of either WT or ΔN of ST6Gal I in culture media was increased compared to increased the levels of the Tyr410-phosphorylated that in control cells after exposure of vector control or Y410 (active) form of p130CAS (p-p130CAS ;Figure4A) ST6Gal I-overexpressing cells to IR (Figure 5C). How- and enhanced cell migration (Figure 4B). Although the ever, even though IR alone slightly increased cell surface ΔN soluble form had no significant effects on sialylation protein sialylation which might be induction of ST6Gal Lee et al. Radiation Oncology 2012, 7:47 Page 6 of 10 http://www.ro-journal.com/content/7/1/47 Figure 3 Sialylation and migration effects of non-cleavable ST6Gal I (L37A/K40A). A. SW480 cells were transfected with WT ST6Gal I (WT) Y410 or ST6Gal I (L37A/K40A) and cell lysates were analyzed for the phosphorylated form of p130CAS (p-P130CAS ) and integrin b1by immunoblotting. Expression of ST6Gal I was assessed using an anti-Flag antibody. Sialylation of integrin b1 was determined by SNA lectin affinity assay. B. SW480 cells were co-transfected with BACE1 and the WT or L37A/K40A form of ST6Gal I. Cell lysates were immunoblotted with antibodies against BACE1 and Flag. C. SW480 cells and CT26 cells were co-transfected with BACE1 and WT or L37A/K40A. Twenty-four hours after transfection, cell migration was determined using Transwell migration assays. Upper: images of migrated cells captured by phase-contrast microscopy; lower: migration measured by counting cells. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). I by IR [23,24], Golg-anchored ST6Gal I-mediated sialylation and migration were partially inhibited by IR, increase of cell surface sialylation was not augmented by an effect that might be due to cleavage of ST6Gal I (Fig- IR (Figure 5D). We also checked the effect of IR on ure 5E). ST6Gal I-induced cell migration. ST6Gal I enhanced CT26 cell migration, an effect that was partially inhib- Discussion ited by IR. These results indicate that IR acted through In the present study, we showed that IR induced clea- induction of BACE1 expression to induce cleavage of vage and secretion of the soluble form of ST6Gal I by ST6Gal I protein to a soluble form that retained sialyl- increasing BACE-1 expression, an effect that may contri- transferase activity. ST6Gal I-induced cell surface bute to IR-induced migration of colon cancer cells, Lee et al. Radiation Oncology 2012, 7:47 Page 7 of 10 http://www.ro-journal.com/content/7/1/47 Figure 4 The soluble form of ST6Gal I increases the migration of colon cancer cells. A. SW480 cells were transfected with WT ST6Gal I Y410 (WT) or ST6Gal I-ΔN(ΔN), and cell lysates were analyzed for the phosphorylated form of p130CAS (p-P130CAS ) and integrin b1by immunoblotting. Sialylation of integrin b1 was determined by SNA lectin affinity assay. B. After transfection with the WT or ΔN form of ST6Gal I, cell migration was determined using Transwell migration assays. Upper: images of migrated cells captured by phase-contrast microscopy; lower: migration measured by counting cells. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). which phenomena is independent of integrin b1sialyla- tissues. Information regarding BACE-1 expression and tion that was usually mediated by Golgi-anchored functions outside of the brain is limited. Furthermore, ST6Gal I. the physiological importance of BACE-1 in cancer pro- Our previous study suggested that IR increases the gression, metastasis, and responsiveness to radiation expression of ST6Gal I, which, in turn, is involved in remains largely unknown. Evidence that BACE-1 is one radioresistance and radiation-induced migration via sia- of the proteases responsible for the cleavage of ST6Gal I lylation of integrin b1 [22,23,26]. Another interesting was provided by experiments employing a b-secretase issue raised previously is that IR increases ST6Gal I inhibitororsiRNA-mediatedknockdown of BACE-1 cleavage and secretion into culture media [23]. While (Figure 1). The results of these experiments suggest that the current data indicate that the Golgi is the main site BACE1 activity involved in cleavage of ST6Gal I from of sialylation by ST6Gal I in most cells, the fact that the Golgi membrane and inducing secretion. soluble forms of ST6Gal I are detected in body fluids Interestingly, inhibition of g-secretase also increased and media from cultured cells has raised questions con- ST6Gal I levels and cell surface sialylation. Although cerning the functions of this extracellular ST6Gal I. It is BACE-1 expression levels could be a major determinant known that ST6Gal I is not the only transferase that of ST6Gal I cleavage, other regulatory mechanisms exists in a soluble form; other glycosyltransferases are might also affect cleavage, secretion, or sorting of also detected in the systemic circulation of cancer ST6Gal I to critical subcellular localizations where patients, where they are associated with disease severity ST6Gal I could encounter proteases. Such BACE-1- and poor prognosis. It has also been suggested that independent mechanisms might also regulate ST6Gal I soluble ST6Gal I may be a potential biomarker for the function. clinical evaluation of colorectal cancer, highlighting the In colon cancer cells, IR increased the expression of importance of elucidating the function of soluble BACE1, which is involved in IR-mediated cleavage of ST6Gal I, particularly in the radiation-induced migration ST6Gal I (Figure 5). To address the detailed mechan- of cancer cells. isms linking ST6Gal I to cancer cell migration, we BACE-1, which is a crucial protease in the pathogen- established non-cleavable (L37A/K40A) and soluble esis of Alzheimer’sdisease,ishighlyexpressed in the (ΔN) variants of ST6Gal I that retained catalytic activity brain, but it is also expressed at low levels in peripheral (Figure 2). Cleaved ST6Gal I was secreted into the Lee et al. Radiation Oncology 2012, 7:47 Page 8 of 10 http://www.ro-journal.com/content/7/1/47 Figure 5 IR-induced BACE-1 mediates cleavage of ST6Gal I, and inhibits sialylation of cell surface molecules and colon cancer cell migration. A. Following irradiation of SW480 and CT26 cells, BACE1 expression levels were determined by RT-PCR and immunoblotting. CT26 vector control cells and cells stably expressing ST6Gal I were exposed to 10 Gy of IR. B. The soluble form of ST6Gal I was analyzed after co- transfection with WT ST6Gal I and BACE1, or after exposure of IR (10 Gy, 48 h) to WT ST6Gal I -transfected cells. C. SW480 and CT26 colorectal carcinoma cell lines were stably transfected with ST6Gal I and exposed to 10 Gy of IR. After 24 hours, culture media were harvested and ST6Gal I activity was assayed by ELISA. Data are presented as means ± SDs from three replicates. CT26 vector control cells and cells stably expressing ST6Gal I were exposed to 10 Gy of IR. After 24, a2,6 sialylation of cell surface was analyzed by flow cytometry. Data are presented as means ± SDs of three replicates. *p < 0.05. n.s., not significant. (D). Cell migration was determined using Transwell migration assays (E). Upper: images of migrated cells captured by phase-contrast microscopy; lower: migration measured by counting cells. Data are presented as means ± SDs of three replicates (*p < 0.05 vs. the corresponding control). Lee et al. Radiation Oncology 2012, 7:47 Page 9 of 10 http://www.ro-journal.com/content/7/1/47 Authors’ contributions culture media after exposure to radiation (Figure 5), and ML and JJP performed experiments. YGK and YSL designed, analyzed data, the truncated soluble ΔN form retained sialyltransferase and wrote paper. All authors have read and approved the final manuscript. activity (Figure 2). We had predicted that the soluble Competing interests form of ST6Gal I was merely a byproduct of the Golgi- The authors declare that they have no competing interests. anchored proform of ST6Gal I that was excreted into the extracellular milieu after exposure of cells to radia- Received: 1 December 2011 Accepted: 27 March 2012 Published: 27 March 2012 tion. However, ST6Gal I-ΔN also induced cellular migration (Figure 4), suggesting that the soluble form of References ST6Gal I could function in tumor cell migration with 1. 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Radiation OncologySpringer Journals

Published: Mar 27, 2012

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