RESUMO
BACKGROUND: Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed. METHODS: Circulating CD14(+)CD16(+) of breast cancer patients and induced CD14(+)CD16(+) DCs were identified as tDCs by treating CD14(+) monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo. RESULTS: The CD14(+)CD16(+) tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c(+) DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c(+)/Ly6G(+)/IL-10(+) in the BALB/c mice. CONCLUSIONS: Galectin-1 can induce tDCs after internalizing into CD14(+) monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy. GENERAL SIGNIFICANCE: Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.
Assuntos
Neoplasias da Mama/imunologia , Células Dendríticas/citologia , Galectina 1/fisiologia , Tolerância Imunológica , Miosina não Muscular Tipo IIA/fisiologia , Animais , Diferenciação Celular , Endocitose , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Mutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors. METHODS: Suppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-ras (val12) up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7-4) harboring the inducible Ha-ras (val12) oncogene, which could be induced by isopropylthio-ß-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters. RESULTS: Retinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-ras (val12) up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells "7-4" by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model. CONCLUSIONS: We confirmed that RbAp46 is a Ha-ras (val12) up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Genes ras , Neoplasias Pulmonares/metabolismo , Regiões Promotoras Genéticas , Proteína 7 de Ligação ao Retinoblastoma/biossíntese , Regulação para Cima , Animais , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Genes ras/fisiologia , Humanos , Neoplasias Pulmonares/patologia , Células MCF-7 , Camundongos , Camundongos Nus , Células NIH 3T3 , Regiões Promotoras Genéticas/fisiologia , Regulação para Cima/fisiologia , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/secundárioRESUMO
The interaction between tumors and their microenvironments leads to a vicious cycle, which strengthens both immune suppression and cancer progression. The present study demonstrates for the first time that tumor-associated dendritic cells (TADCs) are a source of resistin, which is responsible for increasing lung cancer epithelial-to-mesenchymal transition. In addition, large amounts of resistin in the condition medium (CM) of TADCs increase cell migration and invasion, as well as the osteolytic bone metastatic properties of lung cancer cells. Neutralization of resistin from TADC-CM prevents the advanced malignancy-inducing features of TADC-CM. Significantly elevated levels of resistin have been observed in mice transplanted with lung cancer cells, tumor-infiltrating CD11c(+) DCs in human lung cancer samples and lung cancer patients' sera. Induction of lung cancer progression by TADC-derived resistin is associated with increased expression of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), a histone methyltransferase. Resistin-induced WHSC1 increases the dimethylation of histone 3 at lysine 36 and decreases the trimethylation of histone 3 at lysine 27 on the promoter of Twist, resulting in an enhancement of the expression of Twist. Knockdown of WHSC1 by small interfering RNA transfection significantly decreases resistin-mediated cancer progression by decreasing the upregulation of Twist, suggesting that WHSC1 plays a critical role in the regulation of Twist by epigenetic modification. Furthermore, mice that received antiresistin antibodies showed a decreased incidence of cancer development and metastasis. These findings suggest that TADC-derived resistin may be a novel candidate in promoting the development of lung cancer.
Assuntos
Adenocarcinoma/secundário , Carcinoma Pulmonar de Lewis/patologia , Células Dendríticas/patologia , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Repressoras/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Apoptose , Western Blotting , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Estudos de Casos e Controles , Adesão Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Células Dendríticas/metabolismo , Progressão da Doença , Imunofluorescência , Seguimentos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/genética , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Monócitos/patologia , Osteoclastos/metabolismo , Osteoclastos/patologia , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína 1 Relacionada a Twist/antagonistas & inibidores , Proteína 1 Relacionada a Twist/genéticaRESUMO
The interaction between cancer cells and their microenvironment is a vicious cycle that enhances the survival and progression of cancer, resulting in metastasis. This study is the first to indicate that lung cancer-derived galectin-1 secretion is responsible for stimulating tumor-associated dendritic cells (TADCs) production of mature heparin-binding EGF-like growth factor (HB-EGF), which, in turn, increases cancer progression. Treatment of galectin-1, present in large amounts in lung cancer conditioned medium and lung cancer patient sera, mimicked the inductive effect of lung cancer conditioned medium on the expression and ectodomain shedding of HB-EGF by TNFα-converting enzyme/a disintegrin and metalloproteinase 9 (ADAM9) and ADAM17. Significant up-regulation of HB-EGF has been seen in tumor-infiltrating CD11c(+) dendritic cells in human lung cancer samples. Active cleavage of HB-EGF in TADCs by ADAM9 and ADAM17 is associated with increased protein kinase C δ and Lyn signaling. Enhancement of HB-EGF production in TADCs increased the proliferation, migration, and epithelial-to-mesenchymal transition abilities of lung cancer. In contrast, inhibiting HB-EGF by siRNA suppressed TADC-mediated cancer progression. Moreover, mice injected with galectin-1 knockdown Lewis lung carcinoma showed decreased expression and ectodomain shedding of HB-EGF and reduced incidence of cancer development, resulting in increased survival rates. We demonstrate here for the first time that human and mouse DCs are a source of HB-EGF, an EGFR ligand with tumorigenic properties. Antagonists of the effect of lung cancer-derived galectin-1 on DCs and anti-HB-EGF blocking antibodies could, therefore, have therapeutic potential as antitumor agents.
Assuntos
Carcinoma Pulmonar de Lewis/metabolismo , Células Dendríticas/metabolismo , Galectina 1/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Animais , Anticorpos Antineoplásicos/farmacologia , Anticorpos Neutralizantes/farmacologia , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/patologia , Galectina 1/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Metástase Neoplásica , Proteínas de Neoplasias/genética , Proteína Quinase C-delta , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The interaction of cancer within a microenvironment is an important factor determining cancer development. This study analyzed the soluble factors secreted by tumor-associated dendritic cells (TADCs), which are responsible for increasing lung cancer growth, migration, invasion, and epithelial-to-mesenchymal transition. Addition of amphiregulin, present in large amounts in TADC-conditioned medium (CM), mimicked the inductive effect of TADC-CM on lung cancer progression, supported by the enhancement of cell proliferation, migration, and invasion as well as osteolytic bone metastases phenotypes. In contrast, neutralization of amphiregulin from TADC-CM decreased the advanced malignancy-inductive properties of TADC-CM. Significant upregulation of amphiregulin has been seen in tumor-infiltrating CD11c(+) DCs in human lung cancer samples and patients' sera. The enhancement of amphiregulin in TADCs has also been noted in mice transplanted with lung cancer cells. Induction of lung cancer progression by TADC-derived amphiregulin is associated with increased STAT3 and AKT activation, which subsequently increases the expression of cyclin D, Twist, and Snail. Blocking AKT significantly decreases TADC-CM and amphiregulin-mediated migration by decreasing the upregulation of Snail, whereas inhibition of STAT3 reduced the modulation of TADC-derived amphiregulin on Twist and cyclin D expression, suggesting that cooperation of STAT3 and AKT plays a critical role in TADC-mediated cancer progression. Moreover, mice treated with anti-amphiregulin Abs showed decreased incidence of cancer development and increased survival rates. Our study suggests that inhibition of amphiregulin or amphiregulin-related signaling is an attractive therapeutic target in lung cancer patients.
Assuntos
Carcinoma Pulmonar de Lewis/imunologia , Movimento Celular , Proliferação de Células , Células Dendríticas/imunologia , Glicoproteínas/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Neoplasias Pulmonares/imunologia , Anfirregulina , Animais , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Neoplasias Ósseas/terapia , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/terapia , Linhagem Celular Tumoral , Ciclina D/biossíntese , Ciclina D/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Família de Proteínas EGF , Regulação Neoplásica da Expressão Gênica/imunologia , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Proteínas Nucleares/biossíntese , Proteínas Nucleares/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/imunologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/imunologia , Proteína 1 Relacionada a Twist/biossíntese , Proteína 1 Relacionada a Twist/imunologiaRESUMO
Lung cancer, one of the leading causes of death worldwide, is often associated with a state of immune suppression, but the molecular and functional basis remains enigmatic. Evidence is provided in this paper supporting the role of lung cancer-derived soluble lectin, galectin-1, as a culprit in dendritic cell (DC) anergy. We have shown that galectin-1 is highly expressed in lung cancer cell lines, together with the serum and surgical samples from lung cancer patients. Functionally, lung cancer-derived galectin-1 has been shown to alter the phenotypes of monocyte-derived DCs (MdDCs) and impair alloreactive T cell response, concomitant with the increase of CD4(+)CD25(+)FOXP3(+) regulatory T cells. The regulatory effect of galectin-1 is mediated, in part, through its ability to induce, in an Id3 (inhibitor of DNA binding 3)-dependent manner, the expression of IL-10 in monocytes and MdDCs. This effect is inhibited by the addition of lactose, which normalizes the phenotypic and functional alterations seen in MdDCs. Of note, significant upregulation of IL-10 was seen in tumor-infiltrating CD11c(+) DCs in human lung cancer samples. This was also noted in mice transplanted with lung cancer cells, but not in those receiving tumor cells with galectin-1 knockdown. Furthermore, a significant reduction was noted in lung cancer incidence and in the levels of IL-10-expressing, tumor-infiltrating DCs, in mice receiving galectin-1-silenced tumor cells. These results thus suggest that the galectin-1/IL-10 functional axis may be crucial in lung cancer-mediated immune suppression, and that galectin-1 may serve as a target in the development of lung cancer immunotherapy.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/patologia , Galectina 1/fisiologia , Proteínas Inibidoras de Diferenciação/fisiologia , Interleucina-10/fisiologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/imunologia , Animais , Brônquios/imunologia , Brônquios/metabolismo , Brônquios/patologia , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Células Cultivadas , Anergia Clonal/imunologia , Células Dendríticas/metabolismo , Humanos , Imunoterapia Adotiva/métodos , Proteínas Inibidoras de Diferenciação/biossíntese , Interleucina-10/biossíntese , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/biossíntese , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologiaRESUMO
The process of differentiation from monocytes to dendritic cells is critical in immune modulation. Monocyte apoptosis is a key regulator in balancing the immune response. Galectin1 has been reported to induce tolerogenic dendritic cells by the autocrine interleukin (IL)10 in monocytes. However, IL10 has been found to induce apoptosis in IL4/granulocyte macrophage colonystimulating factor (CSF) stimulating and nonstimulating monocytes, whereas galectin1 has not. After analyzing the factors secreted by galectin-1-activated CD14 monocytes isolated from the peripheral blood, the present study revealed that galectin1 upregulates IL10 and granulocyte (G)-CSF expression. Furthermore, GCSF inhibited IL10induced apoptosis, implying that galectin1 may enhance the immunemodulating functions of GCSF by inducing tolerogenic dendritic cells and maintaining their survival. Therefore, GCSF may be further applied in immune therapy, particularly in the IL10presenting microenvironment.
Assuntos
Galectina 1/fisiologia , Fator Estimulador de Colônias de Granulócitos/fisiologia , Interleucina-10/fisiologia , Monócitos/fisiologia , Apoptose , Diferenciação Celular , Células Cultivadas , Células Dendríticas/metabolismo , HumanosRESUMO
Tumor-associated dendritic cells (TADCs) are important in tumor immune surveillance, and it has been reported that the secretion of interleukin (IL)-10 by cancer cells is a major factor involved in the induction of TADCs in the tumor microenvironment. In the present study, IL-10 was found to activate cluster of differentiation (CD)45 protein tyrosine phosphatase (PTPase), inducing a TADC-like phenomenon. The PTPase inhibitor, phenylarsine oxide, and a CD45 inhibitor reversed the IL-10-induced impaired differentiation of the DCs, and also reversed the induction of the TADCs by A549, MDA-MB-231 and SW480 conditioned media, which thus represents a novel therapy to reduce immune surveillance in the tumor microenvironment. The present study is the first to identify that CD45 is involved in IL-10-activated signaling in myeloid lineage cells.
RESUMO
Autophagy plays diverse roles in Ras-related tumorigenesis. H-ras(val12) induces autophagy through multiple signaling pathways including Raf-1/ERK pathway, and various ERK downstream molecules of autophagy have been reported. In this study, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) is identified as a downstream transducer of the Ras/Raf/ERK signaling pathway to induce autophagy. BNIP3 was upregulated by H-ras(val12) at the transcriptional level to compete with Beclin 1 for binding with Bcl-2. H-ras(val12)-induced autophagy suppresses cell proliferation demonstrated both in vitro and in vivo by expression of ectopic BNIP3, Atg5, or interference RNA of BNIP3 (siBNIP3) and Atg5 (shAtg5) using mouse NIH3T3 and embryo fibroblast cells. H-ras(val12) induces different autophagic responses depending on the duration of Ras overexpression. After a short time (48 hours) of Ras overexpression, autophagy inhibits cell proliferation. In contrast, a longer time (2 weeks) of Ras overexpression, cell proliferation was enhanced by autophagy. Furthermore, overexpression of mutant Ras, BNIP3, and LC3-II was detected in bladder cancer T24 cells and the tumor parts of 75% of bladder cancer specimens indicating a positive correlation between autophagy and tumorigenesis. Taken together, our mouse model demonstrates a balance between BNIP3-mediated autophagy and H-ras(val12)-induced tumor formation and reveals that H-ras(val12) induces autophagy in a BNIP3-dependent manner, and the threshold of autophagy plays a decisive role in H-ras(val12)-induced tumorigenesis. Our findings combined with others' reports suggest a new therapeutic strategy against Ras-related tumorigenesis by negative or positive regulation of autophagic activity, which is determined by the level of autophagy and tumor progression stages.