RESUMO
Cancer stem cells are cancer cells characterized with tumor initiating capacity. ß1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) synthesizes GalNAcß1-4GlcNAc (LacdiNAc) which contributes to self-renewal of mouse embryonic stem cells. We previously showed that B4GALNT3 overexpression enhances colon cancer cell malignant phenotypes in vitro and in vivo. However, the role of B4GALNT3 in cancer stemness remains unclear. We found that B4GALNT3 expression was positively correlated with advanced stages and poor survival in colorectal cancer patients. Knockdown of B4GALNT3 using small interfering (si) RNAs in colon cancer cell lines (HCT116, SW480, HCT15, and HT29 cells) decreased sphere formation and the expression of stem cell markers, OCT4 and NANOG. The expression of B4GALNT3 was upregulated in colonospheres. Interestingly, we found that B4GALNT3 primarily modified N-glycans of EGFR with LacdiNAc by Wisteria floribunda agglutinin (WFA) pull down assays. B4GALNT3 knockdown suppressed EGF-induced phosphorylation of EGFR and its downstream signaling molecules. Furthermore, EGF-induced degradation of EGFR was facilitated. In addition, EGF-induced migration and invasion were significantly suppressed by B4GALNT3 knockdown. Taken together, these data suggest B4GALNT3 regulates cancer stemness and the invasive properties of colon cancer cells through modifying EGFR glycosylation and signaling. Our results provide novel insights into the role of LacdiNAc in colorectal cancer development.
Assuntos
Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Receptores ErbB/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Neoplasias do Colo/genética , Técnicas de Silenciamento de Genes , Glicosilação , Células HCT116 , Células HT29 , Humanos , N-Acetilgalactosaminiltransferases/genética , Transdução de SinaisRESUMO
Expression of T antigen (Galbeta1, 3GalNAc) is associated with enhanced metastatic potential and poor prognosis in colorectal cancer. Cosmc is a molecular chaperone required for the formation of an active T-synthase, which catalyzes the synthesis of T antigen. However, the expression and role of Cosmc in colorectal cancer are still unclear. Here, real-time PCR showed that overexpression of Cosmc mRNA in colorectal tumors compared with paired non-tumorous tissues was associated with increased American Joint Committee on Cancer (AJCC) tumor stage. Forced expression of Cosmc in HCT116 cells significantly increased T antigen expression and enhanced cell growth, migration, and invasion, which was associated with increased phosphorylation of focal adhesion kinase (FAK), ERK, and Akt. These Cosmc-enhanced malignant phenotypes were significantly suppressed by specific inhibitor of MEK or PI3K. We also found that Cosmc overexpression increased tumor growth and decreased survival of tumor-bearing SCID mice. Conversely, knockdown of Cosmc with siRNA in SW480 cells decreased malignant behaviors and the signaling pathways, which were substantially reversed by constitutively active Akt or MEK. Taken together, these results suggest that Cosmc promotes malignant phenotypes of colon cancer cells mainly via activation of MEK/ERK and PI3K/Akt signaling pathways, and that Cosmc may serve as a potential target for colorectal cancer treatment.
Assuntos
Neoplasias Colorretais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Chaperonas Moleculares/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Citometria de Fluxo , Quinase 1 de Adesão Focal/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos SCID , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Células Tumorais CultivadasRESUMO
Aberrant expression of the simple mucin-type carbohydrate antigens such as T, Tn, sialyl-T and sialyl-Tn is associated with poor prognosis in several cancers. ß1,3-N-acetylglucosaminyltransferase-3 (B3GNT3), a member of the ß3GlcNAcT family, is responsible for forming extended core 1 (T antigen) oligosaccharides. The role of B3GNT3, which is expressed in various tissues including human fetal brain, in regulating neuroblastoma (NB) formation and cell behaviors remains unclear. Here, we showed that increased B3GNT3 expression evaluated using immunohistochemistry in NB tumor tissues correlated well with the histological grade of differentiation as well as a favorable Shimada's subset of pathology. Univariate and multivariate analyses revealed that positive B3GNT3 expression in tumor tissues predicted a favorable prognosis in NB patients independent of other prognostic markers. B3GNT3 overexpression suppresses T antigen formation and malignant phenotypes including migration and invasion of SK-N-SH cells, whereas B3GNT3 knockdown enhances these phenotypes of SK-N-SH cells. Moreover, B3GNT3 expression decreased phosphorylation of focal adhesion kinase (FAK), Src, paxillin, Akt and ERK1/2. We conclude that B3GNT3 predicts a favorable cancer behavior of NB and suppresses malignant phenotypes by modulating mucin-type O-glycosylation and signaling in NB cells.
Assuntos
Movimento Celular/genética , N-Acetilglucosaminiltransferases/metabolismo , Neuroblastoma/patologia , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glicosilação , Humanos , Masculino , N-Acetilglucosaminiltransferases/biossíntese , N-Acetilglucosaminiltransferases/genética , Invasividade Neoplásica , Neuroblastoma/genética , Neuroblastoma/mortalidade , Paxilina/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Taxa de Sobrevida , Transfecção , Quinases da Família src/metabolismoRESUMO
PURPOSE: Neuroblastoma (NB) is a neural crest-derived tumor that commonly occurs in childhood. ß-1,4-Galactosyltransferase III (B4GALT3) is highly expressed in human fetal brain and is responsible for the generation of poly-N-acetyllactosamine, which plays a critical role in tumor progression. We therefore investigated the expression and role of B4GALT3 in NB. EXPERIMENTAL DESIGN: We examined B4GALT3 expression in tumor specimens from 101 NB patients by immunohistochemistry and analyzed the correlation between B4GALT3 expression and clinicopathologic factors or survival. The functional role of B4GALT3 expression was investigated by overexpression or knockdown of B4GALT3 in NB cells for in vitro and in vivo studies. RESULTS: We found that B4GALT3 expression correlated with advanced clinical stages (P = 0.040), unfavorable Shimada histology (P < 0.001), and lower survival rate (P < 0.001). Multivariate analysis showed that B4GALT3 expression is an independent prognostic factor for poor survival of NB patients. B4GALT3 overexpression increased migration, invasion, and tumor growth of NB cells, whereas B4GALT3 knockdown suppressed the malignant phenotypes of NB cells. Mechanistic investigation showed that B4GALT3-enhanced migration and invasion were significantly suppressed by ß1-integrin blocking antibody. Furthermore, B4GALT3 overexpression increased lactosamine glycans on ß1-integrin, increased expression of mature ß1-integrin via delayed degradation, and enhanced phosphorylation of focal adhesion kinase. Conversely, these properties were decreased by knockdown of B4GALT3 in NB cells. CONCLUSIONS: Our findings suggest that B4GALT3 predicts an unfavorable prognosis for NB and may regulate invasive phenotypes through modulating glycosylation, degradation, and signaling of ß1-integrin in NB cells.
Assuntos
Galactosiltransferases/metabolismo , Integrina beta1/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Pré-Escolar , Feminino , Galactosiltransferases/genética , Expressão Gênica , Glicosilação , Humanos , Lactente , Recém-Nascido , Masculino , Invasividade Neoplásica , Estadiamento de Neoplasias , Neuroblastoma/genética , Neuroblastoma/mortalidade , Fenótipo , Prognóstico , Proteólise , Transdução de SinaisRESUMO
ß1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) promotes the formation of GalNAcß1,4GlcNAc (LacdiNAc or LDN). Drosophila ß1,4-N-acetylgalactosaminyltransferase A (B4GALNTA) contributes to the synthesis of LDN, which helps regulate neuronal development. In this study, we investigated the expression and role of B4GALNT3 in human neuroblastoma (NB). We used IHC analysis to examine 87 NB tumors, and we identified correlations between B4GALNT3 expression and clinicopathologic factors, including patient survival. Effects of recombinant B4GALNT3 on cell behavior and signaling were studied in SK-N-SH and SH-SY5Y NB cells. Increased expression of B4GALNT3 in NB tumors correlated with a favorable histologic profile (P < 0.001, χ² test) and early clinical staging (P = 0.041, χ² test) and was a favorable prognostic factor for survival as evaluated by univariate and multivariate analyses. Reexpression of B4GALNT3 in SK-N-SH and SH-SY5Y cells suppressed cell proliferation, colony formation, migration, and invasion. Moreover, B4GALNT3 increased the LacdiNAc modification of ß1 integrin, leading to decreased phosphorylation of focal adhesion kinase (FAK), Src, paxillin, Akt, and ERK1/2. B4GALNT3-mediated suppression of cell migration and invasion were substantially reversed by concomitant expression of constitutively active Akt or MEK. We conclude that B4GALNT3 predicts a favorable prognosis for NB and suppresses the malignant phenotype via decreasing ß1 integrin signaling.
Assuntos
Biomarcadores Tumorais/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Neuroblastoma/mortalidade , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Sobrevivência Celular , Transformação Celular Neoplásica/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Integrina beta1 , Masculino , Invasividade Neoplásica/prevenção & controle , Neuroblastoma/metabolismo , Prognóstico , Transdução de Sinais , Tretinoína/farmacologiaRESUMO
Mucins play a key role in tumorigenesis. MUC15 is a membrane-bound mucin and the MUC15 messenger RNA (mRNA) has been detected in various organs. However, its role in tumor malignancy is still unclear. This study was to investigate the MUC15 expression in colorectal tumors and the role of MUC15 in colon cancer cells. We found that the mRNA expression of MUC15 was significantly higher in 70.8% (51/72) of colorectal tumors compared with their normal counterparts by real-time reverse transcription-polymerase chain reaction. Immunohistochemistry showed that MUC15 expression was increased in 82.6% (43/52) of colorectal tumors. MUC15 overexpression in HCT116 cells enhanced cell proliferation, cell-extracellular matrix adhesion, colony-forming ability and invasion. Furthermore, these effects were significantly reversed by knockdown of MUC15 with short-hairpin RNA. In nude mice models, MUC15 overexpression significantly (P < 0.01) enhanced tumor growth. In addition, treatment of PD98059 significantly (P < 0.01) inhibited MUC15-enhanced invasion, suggesting that the invasion induced by MUC15 in HCT116 cells was primarily mediated through activation of extracellular signal-regulated kinase 1/2. In conclusion, these results suggest that MUC15 is upregulated in colorectal tumors and its expression enhances the oncogenic potential of colon cancer cells.
Assuntos
Transformação Celular Neoplásica , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mucinas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Animais , Western Blotting , Adesão Celular , Movimento Celular , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Mucinas/antagonistas & inibidores , Mucinas/genética , Invasividade Neoplásica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The enzyme beta1,4-N-acetylgalactosaminyltransferase III (beta4GalNAc-T3) exhibits in vitro activity of synthesizing N,N'-diacetyllactosediamine, GalNAcbeta1,4GlcNAc. Here, we investigate the expression of beta4GalNAc-T3 in primary colon tumors and the effects of its overexpression on HCT116 colon cancer cells. Real-time reverse transcription-PCR showed that the expression of beta4GalNAc-T3 was up-regulated in 72.5% (n = 40) of primary colon tumors compared with their normal counterparts. beta4GalNAc-T3 overexpression resulted in enhanced cell-extracellular matrix adhesion, migration, anchorage-independent cell growth, and invasion of colon cancer cells. Moreover, beta4GalNAc-T3 overexpression increased tumor growth and metastasis and decreased survival of tumor-bearing nude mice. beta4GalNAc-T3 overexpression showed increased tyrosine phosphorylation of focal adhesion kinase and paxillin Y118 as well as increased extracellular signal-regulated kinase phosphorylation. These results suggest that up-regulation of beta4GalNAc-T3 may play a critical role in promoting tumor malignancy and that integrin and mitogen-activated protein kinase signaling pathways could be involved in the underlying mechanism.