RESUMO
The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients with cancer. Previously, we demonstrated that the Ser-322 residue of Trop-2 is phosphorylated by protein kinase Cα (PKCα) and PKCδ. Here, we demonstrate that phosphomimetic Trop-2 expressing cells have markedly decreased E-cadherin mRNA and protein levels. Consistently, mRNA and protein of the E-cadherin-repressing transcription factors zinc finger E-Box binding homeobox 1 (ZEB1) were elevated, suggesting transcriptional regulation of E-cadherin expression. The binding of galectin-3 to Trop-2 enhanced the phosphorylation and subsequent cleavage of Trop-2, followed by intracellular signaling by the resultant C-terminal fragment. Binding of ß-catenin/transcription factor 4 (TCF4) along with the C-terminal fragment of Trop-2 to the ZEB1 promoter upregulated ZEB1 expression. Of note, siRNA-mediated knockdown of ß-catenin and TCF4 increased the expression of E-cadherin through ZEB1 downregulation. Knockdown of Trop-2 in MCF-7 cells and DU145 cells resulted in downregulation of ZEB1 and subsequent upregulation of E-cadherin. Furthermore, wild-type and phosphomimetic Trop-2 but not phosphorylation-blocked Trop-2 were detected in the liver and/or lung of some nude mice bearing primary tumors inoculated intraperitoneally or subcutaneously with wild-type or mutated Trop-2 expressing cells, suggesting that Trop-2 phosphorylation, plays an important role in tumor cell mobility in vivo, too. Together with our previous finding of Trop-2 dependent regulation of claudin-7, we suggest that the Trop-2-mediated cascade involves concurrent derangement of both tight and adherence junctions, which may drive metastasis of epithelial tumor cells.
Assuntos
Galectina 3 , beta Catenina , Animais , Humanos , Camundongos , beta Catenina/genética , beta Catenina/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Galectina 3/genética , Galectina 3/metabolismo , Regulação Neoplásica da Expressão Gênica , Células MCF-7 , Camundongos Nus , RNA Mensageiro/genética , Trofoblastos/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismoRESUMO
Dysfunction of tight junctions is a critical step during the initial stage of tumor progression. Trophoblast cell surface antigen 2 (Trop-2) belongs to the family of tumor-associated calcium signal transducer (TACSTD) and is required for the stability of claudin-7 and claudin-1, which are often dysregulated or lost in carcinogenesis. Here, we investigated the effects of Trop-2 phosphorylation on cell motility. Analyses using HCT116 cells expressing WT Trop-2 (HCT116/WT) or Trop-2 alanine-substituted at Ser-303 (HCT116/S303A) or Ser-322 (HCT116/S322A) revealed that Trop-2 is phosphorylated at Ser-322. Furthermore, coimmunoprecipitation and Transwell assays indicated that Trop-2 S322A interacted with claudin-7 the strongest, and a phosphomimetic variant, Trop-2 S322E, the weakest and that HCT116/S322E cells have the highest motility and HCT116/S322A cells the lowest. All cell lines had similar levels of claudin-7 mRNA, but levels of claudin-7 protein were markedly decreased in the HCT116/S322E cells, suggesting posttranscriptional control of claudin-7. Moreover, claudin-7 was clearly localized to cell-cell borders in HCT116/S322A cells but was diffusely distributed on the membrane and partially localized in the cytoplasm of HCT116/S322E and HCT116/WT cells. These observations suggested that Trop-2 phosphorylation plays a role in the decrease or mislocalization of claudin-7. Using protein kinase C (PKC) inhibitors and PKC-specific siRNAs, we found that PKCα and PKCδ are responsible for Trop-2 phosphorylation. Of note, chemical PKC inhibition and PKCα- and PKCδ-specific siRNAs reduced motility. In summary, our findings provide evidence that Trop-2 is phosphorylated at Ser-322 by PKCα/δ and that this phosphorylation enhances cell motility and decreases claudin-7 localization to cellular borders.
Assuntos
Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Claudinas/genética , Claudinas/metabolismo , Células HCT116 , Humanos , Fosforilação , Ligação ProteicaRESUMO
Both mucin 1 (MUC1) and trophoblast cell surface antigen 2 (Trop-2) are overexpressed in various epithelial tumor cells, and their high expression is correlated with a poor prognosis. Both proteins were expressed in a human breast cancer cell line, MCF-7â¯cells, but neither was in a human colon cancer cell line, HCT116â¯cells. When MUC1 cDNA was introduced into HCT116â¯cells (HCT116/MUC1), expression of Trop-2 was induced. Reciprocally, treatment of MCF-7â¯cells with MUC1 siRNA reduced the level of Trop-2. Mithramycin A, an inhibitor of specificity protein 1 (Sp1) transcription factor, effectively inhibited the expression of Trop-2. Consistently, treatment with Sp1 siRNA reduced the expression of Trop-2. To reveal the relationship between MUC1 and Sp1, coimmunoprecipitation assays were performed. Sp1 was coimmunoprecipitated with MUC1 and the level of coimmunoprecipitated Sp1 increased in relation to the level of induced Trop-2. It is known that galectin-3 is an endogenous ligand of MUC1. Binding of galectin-3 to MUC1 elevated the recruitment of Sp1 to MUC1, and knockdown of galectin-3 reduced the level of Trop-2. These results suggest that the binding of galectin-3 to MUC1 enhances the recruitment of Sp1, leading to promotion of the transcription of Trop-2.
Assuntos
Antígenos de Neoplasias/genética , Moléculas de Adesão Celular/genética , Galectina 3/metabolismo , Mucina-1/metabolismo , Regulação para Cima , Proteínas Sanguíneas , Galectina 3/genética , Galectinas , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Células MCF-7 , Mucina-1/genética , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Fator de Transcrição Sp1/metabolismoRESUMO
Both mucin 1 (MUC1) and galectin-3 are known to be overexpressed in various malignant tumors and associated with a poor prognosis. It has been extensively reported that MUC1 is involved in potentiation of growth factor-dependent signal transduction. Because some carbohydrate moieties carried on MUC1 change to preferable ones for binding of galectin-3 in cancer cells, we speculated that MUC1-mediated signaling may occur through direct binding of galectin-3. Immunochemical studies showed that the distribution of galectin-3 coincided with that of MUC1 in various human tumor tissues but not in human nonmalignant tissues, and the level of galectin-3 retained on the surface of various cancer cells paralleled that of MUC1. Treatment of MUC1-expressing cells with galectin-3 induced phosphorylation of ERK1/2 and Akt following enhanced phosphorylation of MUC1 C-terminal domain, consistently promoting tumor cell malignancy. It is also noted that this enhanced phosphorylation occurred independently of EGF receptor-mediated signaling in both EGF receptor- and MUC1-expressing cells, and multivalency of galectin-3 was important for initiation of MUC1-mediated signaling. Expectedly, both silencing of endogenous galectin-3 and treatment with galectin-3 antagonists down-regulated cell proliferation of MUC1-expressing cells. These results suggest that the binding of galectin-3 to MUC1 plays a key role in MUC1-mediated signaling. Thus, constitutive activation of MUC1-mediated signaling in an autocrine/paracrine manner caused by ligation of galectin-3 promotes uncontrolled tumor cell malignancy. This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated signaling pathway.
Assuntos
Neoplasias do Colo/patologia , Galectina 3/metabolismo , Sistema de Sinalização das MAP Quinases , Mucina-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proliferação de Células , Neoplasias do Colo/enzimologia , Neoplasias do Colo/metabolismo , Células HCT116 , Humanos , Fosforilação , Ligação Proteica , Transdução de SinaisRESUMO
When monocyte-derived immature dendritic cells (imDCs) were stimulated with LPS in the presence of anti-CD33/Siglec-3 mAb, the production of IL-12 and phosphorylation of NF-κB decreased significantly. The cell surface proteins of imDCs were chemically cross-linked, and CD33-linked proteins were analyzed by SDS-PAGE and immunoblotting. It was CD14 that was found to be cross-linked with CD33. A proximity ligation assay also indicated that CD33 was colocalized with CD14 on the cell surface of imDCs. Sialic acid-dependent binding of CD33 to CD14 was confirmed by a plate assay using recombinant CD33 and CD14. Three types of cells (HEK293T cells expressing the LPS receptor complex (Toll-like receptor (TLR) cells), and the LPS receptor complex plus either wild-type CD33 (TLR/CD33WT cells) or mutated CD33 without sialic acid-binding activity (TLR/CD33RA cells)) were prepared, and then the binding and uptake of LPS were investigated. Although the level of LPS bound on the cell surface was similar among these cells, the uptake of LPS was reduced in TLR/CD33WT cells. A higher level of CD14-bound LPS and a lower level of TLR4-bound LPS were detected in TLR/CD33WT cells compared with the other two cell types, probably due to reduced presentation of LPS from CD14 to TLR4. Phosphorylation of NF-κB after stimulation with LPS was also compared. Wild-type CD33 but not mutated CD33 significantly reduced the phosphorylation of NF-κB. These results suggest that CD14 is an endogenous ligand for CD33 and that ligation of CD33 with CD14 modulates with the presentation of LPS from CD14 to TLR4, leading to down-regulation of TLR4-mediated signaling.
Assuntos
Receptores de Lipopolissacarídeos/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Western Blotting , Membrana Celular/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Interleucina-2/metabolismo , Ligantes , Receptores de Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Microscopia Confocal , Modelos Biológicos , Mutação , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Receptor 4 Toll-Like/genéticaRESUMO
Mucin 1 (MUC1) is overexpressed in various human malignant tumors and its expression is correlated with a poor prognosis. MUC1 engages in signal transduction by interacting with receptors for growth and differentiation factors, which contributes to the growth and survival of cancer cells. However, the mechanism by which MUC1 promotes cancer cell invasion remains unclear. Microarray analysis revealed that expression of urokinase-type plasminogen activator (uPA) was elevated in MUC1-overexpressing cells. Furthermore, up- and down-modulation of MUC1 expression was clearly correlated with the change of uPA expression. An immunochemical study showed that the distribution of uPA coincided with that of MUC1 in various human cancer tissues. The MUC1 C-terminal domain (MUC1-CD) was associated with nuclear factor-κB (NF-κB) p65 in MUC1-expressing cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that MUC1-CD existed with NF-κB p65 on the uPA promoter. Luciferase assays indicated that the uPA transcriptional activity was correlated with the level of MUC1 expression and that this MUC1-enhancing effect on the uPA transcription was abolished by introduction of mutations into the NF-κB binding sites on the uPA promoter. These results indicate that formation of the MUC1-CD and NF-κB p65 complex enhanced nuclear translocation of NF-κB p65 and subsequent occupancy of NF-κB binding region on the uPA promoter, leading to elevated transcription of uPA. We also demonstrated that uPA induced by MUC1 enhanced the matrix metalloproteinase (MMP)-2 and -9 activities, and consequently promoted cancer cell invasion. Thus, a MUC1 co-operating NF-κB signaling pathway plays a critical role in cancer cell invasion in MUC1-expressing cells.
Assuntos
Movimento Celular/genética , Mucina-1/genética , Regiões Promotoras Genéticas/genética , Fator de Transcrição RelA/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Dipeptídeos/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Immunoblotting , Imuno-Histoquímica , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Mucina-1/metabolismo , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenilenodiaminas/farmacologia , Ligação Proteica/efeitos dos fármacos , Interferência de RNA , Fator de Transcrição RelA/antagonistas & inibidores , Fator de Transcrição RelA/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
BACKGROUND: Galectin-3 is expressed in a variety of tumors and its expression level is related with tumor progression. Aberrant expression of MUC1 in various tumors is also associated with a poor prognosis. It has been reported that MUC1 is a natural ligand of galectin-3. METHODS: A stable MUC1 transfectant was produced by introducing MUC1 cDNA into mouse 3T3 fibroblasts (MUC1/3T3 cells). MUC1 was prepared from MUC1/3T3 cells; MUC1-N-terminal domain (MUC1-ND) and -C-terminal domain (MUC1-CD) were separated by CsCl ultracentrifugation, and then the galectin-3-binding domain was determined by co-immuniprecipitation assay. After ligation of galectin-3 to 3T3/MUC1 cells, MUC1-CD was immunoprecipitated from the cell lysate. The immunoprecipitate was subjected to SDS-PAGE and Western blotting, followed by detection of co-immunoprecipitated ß-catenin. RESULTS: Galectin-3 binds to the N-terminal domain of MUC1 but not to the C-terminal one. Galectin-3 present on the cell surface increased with the expression of MUC1 and is colocalized with MUC1. It should be noted that ß-catenin was detected in the immunoprecipitate with anti-MUC1-CD Ab from a lysate of galectin-3-treated 3T3/MUC1 cells. CONCLUSIONS: Galectin-3 binds to MUC1-ND and triggers MUC1-mediated signaling in 3T3/MUC1 cells, leading to recruitment of ß-catenin to MUC1-CD. GENERAL SIGNIFICANCE: This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated pathway.
Assuntos
Galectina 3/fisiologia , Mucina-1/fisiologia , beta Catenina/metabolismo , Animais , Camundongos , Mucina-1/química , Células NIH 3T3 , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologiaRESUMO
Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, ß-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of ß-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited ß-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Mucina-1/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células HEK293 , Humanos , Camundongos , Mucina-1/genética , Mucina-1/imunologia , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , beta Catenina/genética , beta Catenina/imunologiaRESUMO
BACKGROUND: Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma. PRINCIPAL FINDINGS: We successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas. CONCLUSION: In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.
Assuntos
Madurella , Micetoma , Micetoma/diagnóstico , Micetoma/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
MUC16/CA125 is over-expressed in human epithelial tumors including ovarian, breast and some other carcinomas. The purpose of this study is to investigate how cell surface MUC16 is functionally involved in tumor progression, with a special focus on the role of its cytoplasmic tail. Forced expression of C-terminal MUC16 fragment (MUC16C) in epithelial cancer cells increased cell migration. We found that MUC16C directly interacted with Src family kinases (SFKs). Notably, localizations of E-cadherin and ß-catenin at the cell-cell contacts were more diffuse in MUC16C transfectants compared with mock transfectants. Furthermore, MUC16C transfectants showed reduced Ca(2+)-dependent cell-cell adhesion, but the treatment of cells with PP2, a SFKs inhibitor, restored this. Because cell surface MUC16 is also associated with the E-cadherin/ß-catenin complex, the over-expression of MUC16 and its interaction with SFKs may enhance SFKs-induced deregulation of E-cadherin. Thus, our results suggest a role for cell surface MUC16 in cell-cell adhesion of epithelial cancer cells.