RESUMO
The ability to distinguish malignant from indolent prostate cancer cells is critically important for identification of clinically significant prostate cancer to minimize unnecessary overtreatment and sufferings endured by patients who have indolent cancer. Recently, we discovered that loss of giantin function as the primary Golgi targeting site for endoplasmic reticulum-derived transport vesicles in aggressive prostate cancer cells caused a shift of the Golgi localization site of α-mannosidase 1A to 130 KDa Golgi matrix protein (GM130)-65 KDa Golgi reassembly-stacking protein (GRASP65) site resulting in emergence of high mannose N-glycans on trans-Golgi enzymes and cell surface glycoproteins. To extend this observation, we isolated two cell clones (Clone 1 and Clone 2) from high passage LNCaP cells, which exhibited androgen refractory property missing in low passage LNCaP cells, and characterized their malignant property. We have found that comparing to Clone 2, which does not have cell surface high mannose N-glycans and exhibits localization of α-mannosidase 1A at giantin site, Clone 1 displays cell surface high mannose N-glycans, exhibits localization of α-mannosidase 1A at GM130-GRASP65 site, and shows a faster rate of closing the wound in a wound healing assay. The results indicate that Golgi localization of α-mannosidase 1A at GM130-GRASP65 site and appearance of cell surface high mannose N-glycans may serve as markers of malignant prostate cancer cells.
Assuntos
Autoantígenos/análise , Complexo de Golgi/patologia , Proteínas da Matriz do Complexo de Golgi/análise , Manose/análise , Proteínas de Membrana/análise , Neoplasias da Próstata/patologia , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Membrana Celular/patologia , Humanos , Masculino , Polissacarídeos/análiseRESUMO
Prostate cancer (PCa) remains the second leading cause of cancer-related deaths in U.S. men due to the development of the castration-resistant (CR) PCa phenotype. A useful cell model for analysis of the molecular mechanism of PCa progression is required for developing targeted therapies toward CR PCa. In this study, we established a PCa cell progressive model in three separate cell lines, of which androgen-independent (AI) cells were derived from respective androgen-sensitive (AS) cells. Those AI PCa cells obtain the biochemical properties of the clinical CR phenotype, including AR and PSA expression as well as enhanced proliferation and tumorigenicity under androgen-deprived conditions. Thus, those AI cells recapitulate CR PCa and exhibit increased oxidant species levels as well as enhanced signaling of proliferation and survival pathways. H2O2 treatment directly enhanced AS cell growth and migration, which was counteracted by antioxidant N-acetyl cysteine (NAC). We further identified p66Shc protein enhances the production of oxidant species which contributes to phenotypic and cell signaling alterations from AS to AI PCa cells. H2O2-treated LNCaP-AS cells had a similar signaling profile to that of LNCaP-AI or p66Shc subclone cells. Conversely, the oxidant species-driven alterations of LNCaP-AI and p66Shc subclone cell signaling is mitigated by p66Shc knockdown. Moreover, LNCaP-AI cells and p66Shc subclones, but not LNCaP-AS cells, develop xenograft tumors with metastatic nodules, correlating with p66Shc protein levels. Together, the data shows that p66Shc enhances oxidant species production that plays a role in promoting PCa progression to the CR stage.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Próstata Resistentes à Castração/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Acetilcisteína/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Peróxido de Hidrogênio/farmacologia , Calicreínas/genética , Calicreínas/metabolismo , Metástase Linfática , Masculino , Camundongos , Camundongos Nus , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismoRESUMO
BACKGROUND: There is a pressing need for biomarkers that can distinguish indolent from aggressive prostate cancer to prevent over-treatment of patients with indolent tumor. METHODS: Golgi targeting of glycosyltransferases was characterized by confocal microscopy after knockdown of GM130, giantin, or both. N-glycans on a trans-Golgi enzyme ß4galactosyltransferase-1 isolated by immunoprecipitation from androgen-sensitive and independent prostate cancer cells were determined by matrix-assisted laser desorption-time of flight-mass spectrometry. In situ proximity ligation assay was employed to determine co-localization of (a) α-mannosidase IA, an enzyme required for processing Man8GlcNAc2 down to Man5GlcNAc2 to enable synthesis of complex-type N-glycans, with giantin, GM130, and GRASP65, and (b) trans-Golgi glycosyltransferases with high mannose N-glycans terminated with α3-mannose. RESULTS: Defective giantin in androgen-independent prostate cancer cells results in a shift of Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65. Consequently, trans-Golgi enzymes and cell surface glycoproteins acquire high mannose N-glycans, which are absent in cells with functional giantin. In situ proximity ligation assays of co-localization of α-mannosidase IA with GM130 and GRASP65, and trans-Golgi glycosyltransferases with high mannose N-glycans are negative in androgen-sensitive LNCaP C-33 cells but positive in androgen-independent LNCaP C-81 and DU145 cells, and LNCaP C-33 cells devoid of giantin. CONCLUSION: In situ proximity ligation assays of Golgi localization of α-mannosidase IA at giantin versus GM130-GRASP65 site, and absence or presence of N-glycans terminated with α3-mannose on trans-Golgi glycosyltransferases may be useful for distinguishing indolent from aggressive prostate cancer cells.
Assuntos
Autoantígenos/genética , Biomarcadores Tumorais/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , alfa-Manosidase/metabolismo , Autoantígenos/metabolismo , Biomarcadores Tumorais/química , Linhagem Celular Tumoral , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Proteínas da Matriz do Complexo de Golgi , Humanos , Masculino , Manose/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Polissacarídeos/biossíntese , Polissacarídeos/química , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Ligação Proteica , Transporte Proteico/genética , alfa-Manosidase/químicaRESUMO
Abnormalities in the Golgi apparatus function are important to the development of alcoholic liver injury. We recently reported that Golgi disorganization in ethanol (EtOH)-treated hepatocytes is caused by impaired dimerization of the largest Golgi matrix protein, giantin. However, little is known about the mechanism which forces fragmentation. Here, in both HepG2 cells overexpressing alcohol dehydrogenase and in rat hepatocytes, we found that EtOH administration reduces the complex between giantin and Rab6a GTPase and results in the S1943 phosphorylation of non-muscle Myosin IIA (NMIIA) heavy chain, thus facilitating NMIIA association with Golgi enzymes, as detected by biochemical approaches and 3D Structured Illumination Microscopy. We revealed that NMIIA-P-S1943 competes with giantin for the Rab6a dimer, which was converted to monomer after Golgi fragmentation. Therefore, Rab6a plays a dual role in the Golgi, serving as master regulator of Golgi organization and disorganization, and that NMIIA and giantin engage in a "tug-of-war". However, the inhibition of F-actin and downregulation of NMIIA or overexpression of NMHC-IIAΔtailpiece, as well the overexpression of dominant negative Rab6a(T27N), preserved a compact Golgi phenotype. Thus, the actomyosin complex forces EtOH-induced Golgi disorganization, and the targeting of NMIIA-P-S1943 may be important for preventing the damaging effects of alcohol metabolism on the cell.
Assuntos
Etanol/farmacologia , Complexo de Golgi/efeitos dos fármacos , Miosina não Muscular Tipo IIA/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Álcool Desidrogenase/metabolismo , Animais , Técnicas de Cultura de Células , Complexo de Golgi/enzimologia , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Masculino , Fosforilação , Ratos WistarRESUMO
The hepatic asialoglycoprotein receptor (ASGP-R) is posttranslationally modified in the Golgi en route to the plasma membrane, where it mediates clearance of desialylated serum glycoproteins. It is known that content of plasma membrane-associated ASGP-R is decreased after ethanol exposure, although the mechanisms remain elusive. Previously, we found that formation of compact Golgi requires dimerization of the largest Golgi matrix protein giantin. We hypothesize that ethanol-impaired giantin function may be related to altered trafficking of ASGP-R. Here we report that in HepG2 cells expressing alcohol dehydrogenase and hepatocytes of ethanol-fed rats, ethanol metabolism results in Golgi disorganization. This process is initiated by dysfunction of SAR1A GTPase followed by altered COPII vesicle formation and impaired Golgi delivery of the protein disulfide isomerase A3 (PDIA3), an enzyme that catalyzes giantin dimerization. Additionally, we show that SAR1A gene silencing in hepatocytes mimics the effect of ethanol: dedimerization of giantin, arresting PDIA3 in the endoplasmic reticulum (ER) and large-scale alterations in Golgi architecture. Ethanol-induced Golgi fission has no effect on ER-to-Golgi transportation of ASGP-R, however, it results in its deposition in cis-medial-, but not trans-Golgi. Thus, alcohol-induced deficiency in COPII vesicle formation predetermines Golgi fragmentation which, in turn, compromises the Golgi-to-plasma membrane transportation of ASGP-R.
Assuntos
Regulação para Baixo , Etanol/farmacologia , Complexo de Golgi/metabolismo , Hepatócitos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Álcool Desidrogenase/metabolismo , Animais , Receptor de Asialoglicoproteína/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Regulação para Baixo/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inativação Gênica/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Proteínas de Membrana/metabolismo , Metaboloma/efeitos dos fármacos , Camundongos , Modelos Biológicos , Fenótipo , Isomerases de Dissulfetos de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , RatosRESUMO
The sialyl Lewis a and x (sLe(a/x)) antigens frequently displayed on the surface of tumor cells are involved in metastasis. Their synthesis has been attributed to altered expression of selective glycosyltransferases. Identification of these glycosyltransferases and the glycoproteins that carry these carbohydrate antigens should help advance our understanding of selectin-mediated cancer metastasis. In this study, quantitative real-time polymerase chain reaction analysis coupled with in situ proximity ligation assay and small interference RNA treatment shows involvement of ß3galactosyltransferase-V in the synthesis of MUC16-associated sLe(a) in H292 cells. Also, α3fucosyltransferase-V, which is absent in BEAS-2B human immortalized bronchial epithelial cells and A549 lung carcinoma cells, participates in the synthesis of MUC1-associated sLe(x) in CFT1 human immortalized bronchial epithelial cells and H292 lung carcinoma cells. Neither selectin ligand is found on MUC1 in BEAS-2B and A549 cells. Knockdown of either enzyme suppresses migration, and selectin tethering and rolling properties of H292 cells under dynamic flow as determined by wound healing and parallel plate flow chamber assays, respectively. These results provide insights into how the synthesis of mucin-associated selectin ligands and the metastatic properties of cancer cells can be regulated by selective glycosyltransferases that work on mucins. They may help develop novel anticancer drugs.
Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Galactosiltransferases/metabolismo , Glicoproteínas de Membrana/metabolismo , Mucinas/metabolismo , Antígeno CA-19-9 , Adesão Celular , Linhagem Celular Tumoral , Células Epiteliais/fisiologia , Humanos , Glicoproteínas de Membrana/genética , Oligossacarídeos/metabolismo , Antígeno Sialil Lewis XRESUMO
Core 2 N-acetylglucosaminyltransferase 2/M (C2GnT-M) synthesizes all three ß6GlcNAc branch structures found in secreted mucins. Loss of C2GnT-M leads to development of colitis and colon cancer. Recently we have shown that C2GnT-M targets the Golgi at the Giantin site and is recycled by binding to non-muscle myosin IIA, a motor protein, via the cytoplasmic tail (CT). But how this enzyme is retained in the Golgi is not known. Proteomics analysis identifies keratin type II cytoskeletal 1 (KRT1) as a protein pulled down with anti-c-Myc antibody or C2GnT-M CT from the lysate of Panc1 cells expressing bC2GnT-M tagged with c-Myc. Yeast two-hybrid analysis shows that the rod domain of KRT1 interacts directly with the WKR(6) motif in the C2GnT-M CT. Knockdown of KRT1 does not affect Golgi morphology but increases the interaction of C2GnT-M with non-muscle myosin IIA and its transportation to the endoplasmic reticulum, ubiquitination, and degradation. During Golgi recovery after brefeldin A treatment, C2GnT-M forms a complex with Giantin before KRT1, demonstrating CT-mediated sequential events of Golgi targeting and retention of C2GnT-M. In HeLa cells transiently expressing C2GnT-M-GFP, knockdown of KRT1 does not affect Golgi morphology but leaves C2GnT-M outside of the Golgi, resulting in the formation of sialyl-T antigen. Interaction of C2GnT-M and KRT1 was also detected in the goblet cells of human colon epithelial tissue and primary culture of colonic epithelial cells. The results indicate that glycosylation and thus the function of glycoconjugates can be regulated by a protein that helps retain a glycosyltransferase in the Golgi.
Assuntos
Complexo de Golgi/metabolismo , Queratina-1/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Transporte Proteico , Animais , Brefeldina A/farmacologia , Citoplasma/genética , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Queratina-1/química , Queratina-1/genética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/químicaRESUMO
UNLABELLED: Prostate cancer progression is associated with upregulation of sialyl-T antigen produced by ß-galactoside α-2,3-sialyltransferase-1 (ST3Gal1) but not with core 2-associated polylactosamine despite expression of core 2 N-acetylglucosaminyltransferase-L (C2GnT-L/GCNT1). This property allows androgen-refractory prostate cancer cells to evade galectin-1 (LGALS1)-induced apoptosis, but the mechanism is not known. We have recently reported that Golgi targeting of glycosyltransferases is mediated by golgins: giantin (GOLGB1) for C2GnT-M (GCNT3) and GM130 (GOLGA2)-GRASP65 (GORASP1) or GM130-giantin for core 1 synthase. Here, we show that for Golgi targeting, C2GnT-L also uses giantin exclusively whereas ST3Gal1 uses either giantin or GM130-GRASP65. In addition, the compact Golgi morphology is detected in both androgen-sensitive prostate cancer and normal prostate cells, but fragmented Golgi and mislocalization of C2GnT-L are found in androgen-refractory cells as well as primary prostate tumors (Gleason grade 2-4). Furthermore, failure of giantin monomers to be phosphorylated and dimerized prevents Golgi from forming compact morphology and C2GnT-L from targeting the Golgi. On the other hand, ST3Gal1 reaches the Golgi by an alternate site, GM130-GRASP65. Interestingly, inhibition or knockdown of non-muscle myosin IIA (MYH9) motor protein frees up Rab6a GTPase to promote phosphorylation of giantin by polo-like kinase 3 (PLK3), which is followed by dimerization of giantin assisted by protein disulfide isomerase A3 (PDIA3), and restoration of compact Golgi morphology and targeting of C2GnT-L. Finally, the Golgi relocation of C2GnT-L in androgen-refractory cells results in their increased susceptibility to galectin-1-induced apoptosis by replacing sialyl-T antigen with polylactosamine. IMPLICATIONS: This study demonstrates the importance of Golgi morphology and regulation of glycosylation and provides insight into how the Golgi influences cancer progression and metastasis.
Assuntos
Galectina 1/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Mucinas/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias da Próstata/patologia , Sialiltransferases/metabolismo , Apoptose , Autoantígenos/metabolismo , Linhagem Celular Tumoral , Dimerização , Glicosilação , Proteínas da Matriz do Complexo de Golgi , Humanos , Masculino , Proteínas de Membrana/química , Fosforilação , Neoplasias da Próstata/metabolismo , Especificidade por Substrato , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
The Golgi apparatus is a highly dynamic organelle which frequently undergoes morphological changes in certain normal physiological processes or in response to stress. The mechanisms are largely not known. We have found that heat shock of Panc1 cells expressing core 2 N-acetylglucosaminyltransferase-M (Panc1-C2GnT-M) induces Golgi disorganization by increasing non-muscle myosin IIA (NMIIA)-C2GnT-M complexes and polyubiquitination and proteasomal degradation of C2GnT-M. These effects are prevented by inhibition or knockdown of NMIIA. Also, the speed of Golgi fragmentation induced by heat shock is found to be positively correlated with the levels of C2GnT-M in the Golgi. The results are reproduced in LNCaP cells expressing high levels of two endogenous glycosyltransferases-core 2 N-acetylglucosaminyltransferase-L:1 and ß-galactoside:α2-3 sialyltransferase 1. Further, during recovery after heat shock, Golgi reassembly as monitored by a Golgi matrix protein giantin precedes the return of C2GnT-M to the Golgi. The results are consistent with the roles of giantin as a building block of the Golgi architecture and a docking site for transport vesicles carrying glycosyltransferases. In addition, inhibition/depletion of HSP70 or HSP90 in Panc1-C2GnT-M cells also causes an increase of NMIIA-C2GnT-M complexes and NMIIA-mediated Golgi fragmentation but results in accumulation or degradation of C2GnT-M, respectively. These results can be explained by the known functions of these two HSP: participation of HSP90 in protein folding and HSP70 in protein folding and degradation. We conclude that NMIIA is the master regulator of Golgi fragmentation induced by heat shock or inhibition/depletion of HSP70/90.
Assuntos
Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Resposta ao Choque Térmico , N-Acetilglucosaminiltransferases/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Sialiltransferases/metabolismo , Linhagem Celular Tumoral , Forma Celular , Citoplasma/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Ligação Proteica , RNA Interferente Pequeno/metabolismo , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Sialyl Lewis antigens are selectin ligands involved in leukocyte trafficking and cancer metastasis. Biosynthesis of these selectin ligands occurs by the sequential actions of several glycosyltransferases in the Golgi apparatus following synthesis of the protein backbone in the endoplasmic reticulum. In this study, we examine how the synthesis of sialyl Lewis a (sLe(a)) is regulated in prostatic cells and identify a mucin that carries this glycotope. We treat human prostatic cells including one normal and three cancerous cells with histone deacetylase inhibitors, valproic acid, tricostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), and then monitor the expression of sLe(a). We have found that SAHA enhances the production of sLe(a) in normal prostatic RWPE-1 cells but not prostatic cancer cells. Employing siRNA technology and co-immunoprecipitation, we show that the sLe(a) is associated with MUC1, which is confirmed by confocal immunofluorescence microscopy and proximity ligation assay. The SAHA-induced production of sLe(a) in RWPE-1 cells is resulted from upregulation of B3GALT1 gene via enhancement of acetylated histone-3 and histone-4. Interestingly, PC3 and LNCaP C-81 cells do not produce detectable amounts of sLe(a) despite expressing high levels of B3GALT1. However, the MUC1-associated sLe(a) is generated in these cells after introduction of MUC1 cDNA. We conclude that the synthesis of sLe(a) is controlled by not only peptide backbone of the glycoprotein but also glycoprotein-specific glycosyltransferases involved in the synthesis of sLe(a). Further, the SAHA induction of this selectin ligand in normal prostatic cells may pose a potentially serious side effect of this drug recently approved by the US Food and Drug Administration.
Assuntos
Mucina-1/biossíntese , Oligossacarídeos/metabolismo , Próstata/metabolismo , Antígeno CA-19-9 , Linhagem Celular , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Glicosiltransferases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histonas/imunologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Masculino , Mucina-1/imunologia , Mucina-1/metabolismo , Oligossacarídeos/imunologia , Próstata/efeitos dos fármacos , Próstata/imunologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Ácido Valproico/farmacologia , VorinostatRESUMO
The Golgi apparatus undergoes morphological changes under stress or malignant transformation, but the precise mechanisms are not known. We recently showed that non-muscle myosin IIA (NMIIA) binds to the cytoplasmic tail of Core 2 N-acetylglucosaminyltransferase mucus-type (C2GnT-M) and transports it to the endoplasmic reticulum for recycling. Here, we report that Golgi fragmentation induced by brefeldin A (BFA) or coatomer protein (ß-COP) knockdown (KD) in Panc1-bC2GnT-M (c-Myc) cells is accompanied by the increased association of NMIIA with C2GnT-M and its degradation by proteasomes. Golgi fragmentation is prevented by inhibition or KD of NMIIA. Using multiple approaches, we have shown that the speed of BFA-induced Golgi fragmentation is positively correlated with the levels of this enzyme in the Golgi. The observation is reproduced in LNCaP cells which express high levels of two endogenous glycosyltransferases--C2GnT-L and ß-galactoside α2,3 sialyltransferase 1. NMIIA is found to form complexes with these two enzymes but not Golgi matrix proteins. The KD of both enzymes or the prevention of Golgi glycosyltransferases from exiting endoplasmic reticulum reduced Golgi-associated NMIIA and decreased the BFA-induced fragmentation. Interestingly, the fragmented Golgi detected in colon cancer HT-29 cells can be restored to a compact morphology after inhibition or KD of NMIIA. The Golgi disorganization induced by the microtubule or actin destructive agent is NMIIA-independent and does not affect the levels of glycosyltransferases. We conclude that NMIIA interacts with Golgi residential but not matrix proteins, and this interaction is responsible for Golgi fragmentation induced by ß-COP KD or BFA treatment. This is a novel non-enzymatic function of Golgi glycosyltransferases.
Assuntos
Complexo de Golgi/enzimologia , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , N-Acetilglucosaminiltransferases/fisiologia , Compostos Benzidrílicos/farmacologia , Brefeldina A/farmacologia , Proteína Coatomer/genética , Proteína Coatomer/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Células HT29 , Proteínas de Choque Térmico/antagonistas & inibidores , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Pirrolidinonas/farmacologia , Sialiltransferases/metabolismo , Tunicamicina/farmacologia , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Core 2 N-acetylglucosaminyltransferase 1 (C2GnT1) is a key enzyme participating in the synthesis of core 2-associated sialyl Lewis x (C2-O-sLe(x)), a ligand involved in selectin-mediated leukocyte trafficking and cancer metastasis. To accomplish that, C2GnT1 needs to be localized to the Golgi and this step requires interaction of its cytoplasmic tail (CT) with a protein that has not been identified. Employing C2GnT1 CT as the bait to perform a yeast two-hybrid screen, we have identified Golgi phosphoprotein 3 (GOLPH3) as a principal candidate protein that interacts with C2GnT1 and demonstrated that C2GnT1 binds to GOLPH3 via the LLRRR(9) sequence in the CT. Confocal fluorescence microscopic analysis shows substantial Golgi co-localization of C2GnT1 and GOLPH3. Upon GOLPH3 knockdown, C2GnT1 is found mainly in the endoplasmic reticulum and decorated with complex-type N-glycans, indicating that the enzyme has been transported to the Golgi but is not retained. Also, we have found that a recombinant protein consisting of C2GnT1 CT(1-16)-Leu(17-32)-Gly(33-42)-GFP is localized to the Golgi although the same construct with mutated CT (AAAAA(9)) is not. The data demonstrate that the C2GnT1 CT is necessary and sufficient for Golgi localization of C2GnT1. Furthermore, GOLPH3 knockdown results in reduced synthesis of C2-O-sLe(x) associated with P-selectin glycoprotein ligand-1, reduced cell tethering to and rolling on immobilized P- or E-selectin, and compromised E-selectin-induced activation of spleen tyrosine kinase and cell adhesion to intercellular adhesion molecule-1 under dynamic flow. Our results reveal that GOLPH3 can regulate cell-cell interaction by controlling Golgi retention of C2GnT1.
Assuntos
Comunicação Celular/fisiologia , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Motivos de Aminoácidos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , N-Acetilglucosaminiltransferases/genética , Ligação Proteica , Transporte Proteico/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Quinase SykRESUMO
Glycosylation of secreted and membrane-bound mucins is carried out by glycosyltransferases localized to specific Golgi compartments according to the step in which each enzyme participates. However, the Golgi-targeting mechanisms of these enzymes are not clear. Herein, we investigate the Golgi-targeting mechanisms of core 1 ß3 galactosyltransferase (C1GalT1) and core 2 ß1,6-N-acetylglucosaminyltransferase-2 or mucus type (C2GnT-M), which participate in the early O-glycosylation steps. siRNAs, co-immunoprecipitation, and confocal fluorescence microscopy were employed to identify the golgins involved in the Golgi docking of vesicular complexes (VCs) that carry these two enzymes. We have found that these VCs use different golgins for docking: C2GnT-M-carrying VC (C2GnT-M-VC) utilizes Giantin, whereas C1GalT1-VC employs GM130-GRASP65 complex. However, in the absence of GRASP65, C1GalT1-VC utilizes GM130-Giantin complex. Also, we have found that these VCs are 1.1-1.2 µm in diameter, specific for each enzyme, and independent of coat protein complex II and I (COPII and COPI). These two fluorescently tagged enzymes exhibit different fluorescence recovery times in the Golgi after photobleaching. Thus, novel enzyme-specific Golgi-targeting mechanisms are employed by glycosyltransferases, and multiple Golgi docking strategies are utilized by C1GalT1.
Assuntos
Galactosiltransferases/metabolismo , Complexo de Golgi/enzimologia , N-Acetilglucosaminiltransferases/metabolismo , Vesículas Transportadoras/enzimologia , Autoantígenos/genética , Autoantígenos/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proteína Coatomer/genética , Proteína Coatomer/metabolismo , Galactosiltransferases/genética , Glicosilação , Proteínas da Matriz do Complexo de Golgi , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , N-Acetilglucosaminiltransferases/genética , Ligação Proteica , Transporte Proteico , Interferência de RNA , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína Vermelha FluorescenteRESUMO
Altered glycosylation is a universal feature of cancer cells and altered glycans can help cancer cells escape immune surveillance, facilitate tumor invasion, and increase malignancy. The goal of this study was to identify specific glycoenzymes, which could distinguish prostate cancer cells from normal prostatic cells. We investigated enzymatic activities and gene expression levels of key glycosyl- and sulfotransferases responsible for the assembly of O- and N-glycans in several prostatic cells. These cells included immortalized RWPE-1 cells derived from normal prostatic tissues, and prostate cancer cells derived from metastasis in bone (PC-3), brain (DU145), lymph node (LNCaP), and vertebra (VCaP). We found that all cells were capable of synthesizing complex N-glycans and O-glycans with the core 1 structure, and each cell line had characteristic biosynthetic pathways to modify these structures. The in vitro measured activities corresponded well to the mRNA levels of glycosyltransferases and sulfotransferases. Lectin and antibody binding to whole cells supported these results, which form the basis for the development of tumor cell-specific targeting strategies.
Assuntos
Glicoproteínas/metabolismo , Glicosiltransferases/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Sulfotransferases/metabolismo , Células CACO-2 , Glicoproteínas/genética , Glicosilação , Glicosiltransferases/genética , Humanos , Masculino , Metástase Neoplásica , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Sulfotransferases/genéticaRESUMO
The mechanism of the Golgi-to-ER transport of Golgi glycosyltransferases is not clear. We utilize a cell line expressing the core 2 N-acetylglucosaminyltransferase-M (C2GnT-M) tagged with c-Myc to explore this mechanism. By immunoprecipitation using anti-c-Myc antibodies coupled with proteomics analysis, we have identified several proteins including non-muscle myosin IIA (NMIIA), heat shock protein (HSP)-70 and ubiquitin activating enzyme E1 in the immunoprecipitate. Employing yeast-two-hybrid analysis and pulldown experiments, we show that the C-terminal region of the NMIIA heavy chain binds to the 1-6 amino acids in the cytoplasmic tail of C2GnT-M. We have found that NMIIA co-localizes with C2GnT-M at the periphery of the Golgi. In addition, inhibition or knockdown of NMIIA prevents the brefeldin A-induced collapse of the Golgi as shown by the inhibition of the migration of both Giantin, a Golgi matrix protein, and C2GnT-M, a Golgi non-matrix protein, to the ER. In contrast, knockdown of HSP70 retains Giantin in the Golgi but moves C2GnT-M to the ER, a process also blocked by inhibition or knockdown of NMIIA. Also, the intracellular distribution of C2GnT-M is not affected by knockdown of ß-coatomer protein with or without inhibition of HSPs, suggesting that the Golgi-to-ER trafficking of C2GnT-M does not depend on coat protein complex-I. Further, inhibition of proteasome results in accumulation of ubiquitinated C2GnT-M, suggesting its degradation by proteasome. Therefore, NMIIA and not coat protein complex-I is responsible for transporting the Golgi glycosyltransferase to the ER for proteasomal degradation. The data suggest that NMIIA is involved in the Golgi remodeling.
Assuntos
Retículo Endoplasmático/metabolismo , Glicosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Miosina não Muscular Tipo IIA/metabolismo , Sequência de Aminoácidos , Animais , Técnicas de Cultura de Células , Linhagem Celular , Citoplasma/enzimologia , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Retículo Endoplasmático/enzimologia , Complexo de Golgi/metabolismo , Humanos , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Transporte Proteico , TransfecçãoRESUMO
Sialyl Lewis x (sLe(x)) plays an important role in cancer metastasis. But, the mechanism for its production in metastatic cancers remains unclear. The objective of current study was to examine the effects of a proinflammatory cytokine on the expression of glycosyltransferase and sulfotransferase genes involved in the synthesis of selectin ligands in a prostate cancer cell line. Androgen-independent human lymph node-derived metastatic prostate cancer cells (C-81 LNCaP), which express functional androgen receptor and mimic the castration-resistant advanced prostate cancer, were used. TNFα treatment of these cells increased their binding to P-, E- and L-selectins, anti-sLe(x) antibody, and anti-6-sulfo-sialyl Lewis x antibody by 12%, 240%, 43%, 248% and 21%, respectively. Also, the expression of C2GnT-1, B4GalT1, GlcNAc6ST3, and ST3Gal3 genes was significantly upregulated. Further treatment of TNFα-treated cells with either anti-sLe(x) antibody or E-selectin significantly suppressed their in vitro migration (81% and 52%, respectively) and invasion (45% and 56%, respectively). Our data indicate that TNFα treatment enhances the motility and invasion properties of LNCaP C-81 cells by increasing the formation of selectin ligands through stimulation of the expression of selective glycosyl- and sulfotransferase genes. These results support the hypothesis that inflammation contributes to cancer metastasis.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicosiltransferases/genética , Oligossacarídeos/biossíntese , Neoplasias da Próstata/patologia , Sulfotransferases/genética , Fator de Necrose Tumoral alfa/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Selectina E/metabolismo , Humanos , Selectina L/metabolismo , Ligantes , Masculino , Invasividade Neoplásica , Selectina-P/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Antígeno Sialil Lewis X , Fator de Necrose Tumoral alfa/farmacologiaAssuntos
Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Mucinas/química , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/química , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/classificação , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Neoplasias Colorretais/genética , Cricetinae , Ciclo Estral/genética , Feminino , Regulação da Expressão Gênica , Humanos , Mesocricetus , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/classificação , N-Acetilglucosaminiltransferases/genética , Invasividade Neoplásica , Filogenia , Sequências de Repetição em TandemRESUMO
BACKGROUND: p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism. METHODS AND FINDINGS: In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins. CONCLUSIONS: The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.
Assuntos
Proliferação de Células/efeitos dos fármacos , Neoplasias Ovarianas/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Esteroides/farmacologia , Ubiquitinação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Neoplasias Ovarianas/química , Neoplasias Ovarianas/patologia , Neoplasias da Próstata/química , Neoplasias da Próstata/patologia , Receptores de Esteroides/fisiologia , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
Sialyl Lewis X is a tumor-associated antigen frequently found in the advanced cancers. However, the mechanism for the production of this cancer antigen is not entirely clear. The objective of this study is to examine whether epigenetics is involved in the regulation of the formation of this antigen. We observed an increase of sialyl Lewis X in HCT15 cells, a colon cancer cell line, treated with 5-Aza-2'-deoxycytidine. This treatment enhanced the expression of ß-galactoside:α2,3-sialyltransferase 6 gene and sialyl Lewis X on MUC1, and the adherence of these cells to E-selectin under dynamic flow conditions. In addition, 5-Aza-2'-deoxycytidine treatment inhibited methylation of ß-galactoside:α2,3-sialyltransferase 6 gene and siRNA knockdown of this gene drastically reduced sialyl Lewis X without affecting MUC1 expression. We conclude that 5-Aza-2'-deoxycytidine treatment increases sialyl Lewis X on MUC1 by stimulating the ß-galactoside:α2,3-sialyltransferase 6 gene via inhibition of DNA methylation. Increased sialyl Lewis X by 5-Aza-2'-deoxycytidine raises a concern about the safety of this chemotherapeutic drug. In addition, ß-galactoside:α2,3-sialyltransferase 6 gene may be a potential therapeutic target for suppressing tumorigenicity of colon cancer.
Assuntos
Azacitidina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Mucina-1/metabolismo , Oligossacarídeos/metabolismo , Sialiltransferases/genética , Azacitidina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Decitabina , Selectina E/metabolismo , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicogênio/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Proteínas Imobilizadas/metabolismo , Peso Molecular , Oligossacarídeos/biossíntese , Oligossacarídeos/química , Antígeno Sialil Lewis X , Ativação Transcricional/efeitos dos fármacos , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Human prostate cancer LNCaP cells including C-33 and C-81 cells were originally derived from the lymph nodes of a patient with metastatic prostate cancer. These two cells were employed for characterization of L-selectin ligand and in vitro tumorigenicity, because they mimic the clinical conditions of early and late-stage human prostate cancer. C-81 cells exhibit higher in vitro migratory and invasive properties as compared with C-33 cells. We find that the L-selectin ligand and mucin glycan-associated MECA-79 epitope were elevated in C-81 cells. An increase of these glycotopes positively correlates with elevated tumorigenicity and expression of key glycosyl- and sulfotransferase genes. These results suggest that modulated expression of selective glycogenes correlates with altered tumorigenicity of cancer cells.