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1.
Cancer Cell Int ; 23(1): 165, 2023 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-37568162

RESUMO

BACKGROUND: Breast malignancies are the predominant cancer-related cause of death in women. New methods of diagnosis, prognosis and treatment are necessary. Previously, we identified the breast cancer cell surface protein ADAM8 as a marker of poor survival, and a driver of Triple-Negative Breast Cancer (TNBC) growth and spread. Immunohistochemistry (IHC) with a research-only anti-ADAM8 antibody revealed 34.0% of TNBCs (17/50) expressed ADAM8. To identify those patients who could benefit from future ADAM8-based interventions, new clinical tests are needed. Here, we report on the preclinical development of a highly specific IHC assay for detection of ADAM8-positive breast tumors. METHODS: Formalin-fixed paraffin-embedded sections of ADAM8-positive breast cell lines and patient-derived xenograft tumors were used in IHC to identify a lead antibody, appropriate staining conditions and controls. Patient breast cancer samples (n = 490) were used to validate the assay. Cox proportional hazards models assessed association between survival and ADAM8 expression. RESULTS: ADAM8 staining conditions were optimized, a lead anti-human ADAM8 monoclonal IHC antibody (ADP2) identified, and a breast staining/scoring control cell line microarray (CCM) generated expressing a range of ADAM8 levels. Assay specificity, reproducibility, and appropriateness of the CCM for scoring tumor samples were demonstrated. Consistent with earlier findings, 36.1% (22/61) of patient TNBCs expressed ADAM8. Overall, 33.9% (166/490) of the breast cancer population was ADAM8-positive, including Hormone Receptor (HR) and Human Epidermal Growth Factor Receptor-2 (HER2) positive cancers, which were tested for the first time. For the most prevalent HR-positive/HER2-negative subtype, high ADAM8 expression identified patients at risk of poor survival. CONCLUSIONS: Our studies show ADAM8 is widely expressed in breast cancer and provide support for both a diagnostic and prognostic value of the ADP2 IHC assay. As ADAM8 has been implicated in multiple solid malignancies, continued development of this assay may have broad impact on cancer management.

2.
Breast Cancer Res ; 18(1): 40, 2016 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-27039296

RESUMO

BACKGROUND: ADAM8 (a disintegrin and metalloproteinase 8) protein promotes the invasive and metastatic phenotype of triple-negative breast cancer (TNBC) cells. High ADAM8 expression in breast cancer patients is an independent predictor of poor prognosis. Here, we investigated whether ADAM8 regulates specific miRNAs, their roles in aggressive phenotype, and potential use as biomarkers of disease. METHODS: Microarray analysis was performed on RNA from MDA-MB-231 cells after transient ADAM8 knockdown using TaqMan miRNA cards. Changes in miRNA levels were confirmed using two ADAM8 siRNAs in TNBC cell lines. Kinase inhibitors, ß1-integrin antagonist antibody, and different forms of ADAM8 were employed to elucidate the signaling pathway required for miR-720 expression. miR-720 levels were modulated using a specific antagomiR or a mimic, and effects on aggressive phenotype of TNBC cells were determined using Boyden chamber and 3D-Matrigel outgrowth assays. Plasma was isolated from mice before and after implantation of MDA-MB-231 cells and analyzed for miR-720 levels. Serum samples of TNBC patients were evaluated for their ADAM8 and miR-720 levels. RESULTS: We identified 68 miRNAs differentially regulated upon ADAM8 knockdown, including decreased levels of secreted miR-720. Ectopic overexpression of wild-type ADAM8 or forms that lack metalloproteinase activity similarly induced miR-720 levels. The disintegrin and cysteine-rich domains of ADAM8 were shown to induce miR-720 via activation of a ß1-integrin to ERK signaling cascade. Knockdown of miR-720 led to a significant decrease in migratory and invasive abilities of TNBC cells. Conversely, miR-720 overexpression rescued these properties. A profound increase in plasma levels of miR-720 was detected 7 days after TNBC cell inoculation into mouse mammary fat pads when tumors were barely palpable. Concordantly, miR-720 levels were found to be significantly higher in serum samples of TNBC patients with high ADAM8 expression. CONCLUSIONS: We have shown for the first time that miR-720 is induced by ADAM8 signaling via ERK and plays an essential role in promoting the aggressive phenotype of TNBCs. miR-720 is elevated in serum of patients with ADAM8-high TNBC and, in a group with other miRNAs downstream of ADAM8, holds promise as a biomarker for early detection of or treatment response of ADAM8-positive TNBCs.


Assuntos
Proteínas ADAM/biossíntese , Proteínas de Membrana/biossíntese , MicroRNAs/biossíntese , Invasividade Neoplásica/genética , Neoplasias de Mama Triplo Negativas/genética , Proteínas ADAM/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/genética , Camundongos , MicroRNAs/genética , RNA Interferente Pequeno , Neoplasias de Mama Triplo Negativas/patologia
3.
Nat Cell Biol ; 9(4): 470-8, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17369819

RESUMO

Aberrant constitutive expression of c-Rel, p65 and p50 NF-kappaB subunits has been reported in over 90% of breast cancers. Recently, we characterized a de novo RelB NF-kappaB subunit synthesis pathway, induced by the cytomegalovirus (CMV) IE1 protein, in which binding of p50-p65 NF-kappaB and c-Jun-Fra-2 AP-1 complexes to the RELB promoter work in synergy to potently activate transcription. Although RelB complexes were observed in mouse mammary tumours induced by either ectopic c-Rel expression or carcinogen exposure, little is known about RelB in human breast disease. Here, we demonstrate constitutive de novo RelB synthesis is selectively active in invasive oestrogen receptor alpha (ERalpha)-negative breast cancer cells. ERalpha signalling reduced levels of functional NF-kappaB and Fra-2 AP-1 and inhibited de novo RelB synthesis, leading to an inverse correlation between RELB and ERalpha gene expression in human breast cancer tissues and cell lines. Induction of Bcl-2 by RelB promoted the more invasive phenotype of ERalpha-negative cancer cells. Thus, inhibition of de novo RelB synthesis represents a new mechanism whereby ERalpha controls epithelial to mesenchymal transition (EMT).


Assuntos
Estrogênios/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Transdução de Sinais/fisiologia , Fator de Transcrição RelB/genética , Animais , Northern Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Transformação Celular Neoplásica/genética , Ensaio de Desvio de Mobilidade Eletroforética , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/fisiologia , Feminino , Antígeno 2 Relacionado a Fos/genética , Antígeno 2 Relacionado a Fos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Camundongos , Microscopia de Fluorescência , NF-kappa B/genética , NF-kappa B/metabolismo , Fenótipo , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelB/metabolismo
4.
Pharmaceutics ; 16(4)2024 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-38675197

RESUMO

New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange-mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment.

5.
J Cell Physiol ; 220(3): 593-9, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19373868

RESUMO

Aberrant constitutive expression of the NF-kappaB c-Rel and RelA subunits in breast cancer cells was shown to promote their survival. Recently, we demonstrated that aggressive breast cancers constitutively express high levels of the RelB subunit, which promotes their more invasive phenotype via induction of the BCL2 gene. As these cancers are frequently resistant to therapy, here we tested the hypothesis that RelB promotes their survival. High RelB expressing Hs578T and MDA-MB-231 breast cancer cells were more resistant to gamma-radiation than MCF7 and ZR-75 cells, which express lower RelB levels. Knockdown of RelB in Hs578T led to decreased survival in response to gamma-irradiation, while conversely ectopic expression of RelB in MCF7 cells protected these cells from radiation. Similar data were obtained upon treatment of Hs578T or MCF7 cells with the chemotherapeutic agent doxorubicin. High serum levels of 25-hydroxyvitamin D are associated with decreased breast cancer risk and mortality, although, the mechanisms of its protective actions have not been fully elucidated. Treatment of Hs578T and Her-2/neu-driven NF639 cells with 1,25-dihydroxyvitamin D3 decreased RelB/RELB gene expression and levels of pro-survival targets Survivin, MnSOD and Bcl-2, while increasing their sensitivity to gamma-irradiation. Thus, RelB, which promotes survival and a more highly invasive phenotype of breast cancer cells, is a target of 1,25-dihydroxyvitamin D3, providing one mechanism for the observed protective role of 25-hydroxyvitamin D in patients with breast cancer.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Calcitriol/farmacologia , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Tolerância a Radiação , Radiossensibilizantes/farmacologia , Fator de Transcrição RelB/antagonistas & inibidores , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimioterapia Adjuvante , Relação Dose-Resposta à Radiação , Feminino , Humanos , Proteínas Inibidoras de Apoptose , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , Superóxido Dismutase/metabolismo , Survivina , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo , Transfecção
6.
J Cell Biochem ; 104(6): 2091-106, 2008 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-18393360

RESUMO

The p27(Kip1) (p27) cyclin-dependent kinase inhibitor and c-Myc oncoprotein play essential roles in control of cell cycle progression and apoptosis. Induction of p27 (CDKN1B) gene transcription by Forkhead box O proteins such as FOXO3a leads to growth arrest and apoptosis. Previously, we observed that B cell receptor (surface IgM) engagement of WEHI 231 immature B lymphoma cells with an anti-IgM antibody results in activation of FOXO3a, growth arrest and apoptosis. As ectopic c-Myc expression in these cells prevented anti-IgM induction of p27 and cell death, we hypothesized that c-Myc represses FOXO3a-mediated transcription. Here we show that c-Myc inhibits FOXO3a-mediated activation of the p27 promoter in multiple cell lines. The mechanism of this repression was explored using a combination of co-immunoprecipitation, oligonucleotide precipitation, and chromatin immunoprecipitation experiments. The studies demonstrate a functional association of FOXO3a and c-Myc on a proximal Forkhead binding element in the p27 promoter. This association involves the Myc box II domain of c-Myc and the N-terminal DNA-binding portion of FOXO3a. Analysis of publicly available microarray datasets showed an inverse pattern of c-MYC and p27 RNA expression in primary acute myeloid leukemia, prostate cancer and tongue squamous cell carcinoma samples. The inhibition of FOXO3a-mediated activation of the p27 gene by the high aberrant expression of c-Myc in many tumor cells likely contributes to their uncontrolled proliferation and invasive phenotype.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Animais , Apoptose , Sítios de Ligação , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/química , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias/enzimologia , Neoplasias/patologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Prostate ; 67(1): 83-106, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17013881

RESUMO

BACKGROUND: A major focus of prostate cancer research has been to identify genes that are deregulated during tumor progression, potentially providing diagnostic markers and therapeutic targets. METHODS: We have employed serial analysis of gene expression (SAGE) and microarray hybridization to identify alterations that occur during malignant transformation in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. Many of these alterations were validated by real-time PCR (rtPCR). RESULTS: We identified several hundred mRNAs that were deregulated. Cluster analysis of microarray profiles with samples from various stages of the disease demonstrated that androgen-independent (AI) primary tumors are similar to metastases; 180 transcripts have expression patterns suggesting an involvement in the genesis of late-stage tumors, and our data support a role for phospholipase A2 group IIA in the acquisition of their highly aggressive characteristics. CONCLUSIONS: Our analyses identified well-characterized genes that were previously known to be involved in prostate cancer, validating our study, and also uncovered transcripts that had not previously been implicated in prostate cancer progression.


Assuntos
Adenocarcinoma/genética , Androgênios/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Genes Neoplásicos/fisiologia , Engenharia Genética/métodos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/genética , Adenocarcinoma/metabolismo , Androgênios/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Neoplasias da Próstata/metabolismo , Especificidade da Espécie
8.
Biomed Opt Express ; 7(3): 1042-50, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-27231606

RESUMO

Circulating tumor cell clusters (CTCCs) are significantly more likely to form metastases than single tumor cells. We demonstrate the potential of backscatter-based flow cytometry (BSFC) to detect unique light scattering signatures of CTCCs in the blood of mice orthotopically implanted with breast cancer cells and treated with an anti-ADAM8 or a control antibody. Based on scattering detected at 405, 488, and 633 nm from blood samples flowing through microfluidic devices, we identified 14 CTCCs with large scattering peak widths and intensities, whose presence correlated strongly with metastasis. These initial studies demonstrate the potential to detect CTCCs via label-free BSFC.

9.
EMBO Mol Med ; 6(2): 278-94, 2014 02.
Artigo em Inglês | MEDLINE | ID: mdl-24375628

RESUMO

The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via ß1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.


Assuntos
Proteínas ADAM/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Membrana/metabolismo , Proteínas ADAM/química , Animais , Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/irrigação sanguínea , Adesão Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Integrina beta1/metabolismo , Proteínas de Membrana/química , Camundongos , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Neovascularização Patológica/patologia , Fenótipo , Prognóstico , Estrutura Terciária de Proteína , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais Cultivadas
10.
PLoS One ; 8(9): e73464, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039951

RESUMO

Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by high rates of proliferation, lymphangiogenesis and metastasis, and an overall poor survival. As regular green tea consumption has been associated with improved prognosis of breast cancer patients, including decreased risk of recurrence, here the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) were tested on two IBC lines: SUM-149 and SUM-190. EGCG decreased expression of genes that promote proliferation, migration, invasion, and survival. Consistently, growth, invasive properties, and survival of IBC cells were reduced by EGCG treatment. EGCG also reduced lymphangiogenesis-promoting genes, in particular VEGF-D. Conditioned media from EGCG-treated IBC cells displayed decreased VEGF-D secretion and reduced ability to promote lymphangiogenesis in vitro as measured by hTERT-HDLEC lymphatic endothelial cell migration and tube formation. Tumorsphere formation by SUM-149 cells was robustly inhibited by EGCG, suggesting effects on self-renewal ability. Stem-like SUM-149 cells with high aldehyde dehydrogenase (ALDH) activity, previously implicated in poor patient prognosis, were isolated. EGCG treatment reduced growth and induced apoptosis of the stem-like SUM-149 cells in culture. In an orthotopic mouse model, EGCG decreased growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells and their expression of VEGF-D, which correlated with a significant decrease in peritumoral lymphatic vessel density. Thus, EGCG inhibits the overall aggressive IBC phenotype. Reduction of the stem-like cell compartment by EGCG may explain the decreased risk of breast cancer recurrence among green tea drinkers. Recent clinical trials demonstrate the efficacy of green tea polyphenol extracts in treatment of prostate cancer and lymphocytic leukemia with low toxicity. Given the poor prognosis of IBC patients, our findings suggest further exploration of EGCG or green tea in combinatorial treatments against active IBC disease or in maintenance regimens to avoid recurrence is warranted.


Assuntos
Anticarcinógenos/uso terapêutico , Mama/efeitos dos fármacos , Mama/patologia , Catequina/análogos & derivados , Neoplasias Inflamatórias Mamárias/tratamento farmacológico , Neoplasias Inflamatórias Mamárias/patologia , Animais , Anticarcinógenos/farmacologia , Mama/metabolismo , Catequina/farmacologia , Catequina/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/metabolismo , Linfangiogênese/efeitos dos fármacos , Camundongos , Camundongos SCID , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Fator D de Crescimento do Endotélio Vascular/metabolismo
11.
Mol Cell Biol ; 29(14): 3832-44, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19433448

RESUMO

Aberrant constitutive expression of NF-kappaB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERalpha)-negative breast cancers versus ERalpha-positive ones, due in part to repression of RelB synthesis by ERalpha signaling. Notably, RelB promoted a more invasive phenotype in ERalpha-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERalpha synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERalpha (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERalpha-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-kappaB subunit mediates repression, specifically of ERalpha, thereby promoting a more migratory phenotype.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Proteínas Repressoras/biossíntese , Fator de Transcrição RelB/metabolismo , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Primers do DNA/genética , Receptor alfa de Estrogênio/genética , Feminino , Expressão Gênica , Genes bcl-2 , Humanos , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Fenótipo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas ras/metabolismo
12.
J Biol Chem ; 282(24): 17475-85, 2007 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-17446175

RESUMO

CD40, a tumor necrosis factor receptor family member, is expressed on B lymphocytes. Interaction between CD40 and its ligand (CD40L), expressed on activated T lymphocytes, is critical for B cell survival. Here, we demonstrate that CD40 signals B cell survival in part via transcriptional activation of the RelB NF-kappaB subunit. CD40L treatment of chronic lymphocytic leukemia cells induced levels of relB mRNA. Similarly, CD40L-mediated rescue of WEHI 231 B lymphoma cells from apoptosis induced upon B cell receptor (surface IgM) engagement led to increased relB mRNA levels. Recently, we characterized a new de novo synthesis pathway for the RelB NF-kappaB subunit, induced by the cytomegalovirus IE1 protein, in which binding of p50/p65 NF-kappaB and c-Jun/Fra-2 AP-1 complexes to the relB promoter works in synergy to potently activate transcription (Wang, X., and Sonenshein, G. E. (2005) J. Virol. 79, 95-105). CD40L treatment of WEHI 231 cells caused induction of AP-1 family members Fra-2, c-Jun, JunD, and JunB. Cotransfection of Fra-2 with the Jun AP-1 subunits and p50/c-Rel NF-kappaB led to synergistic activation of the relB promoter. Ectopic expression of relB or RelB knockdown using small interfering RNA demonstrated the important role of this subunit in control of WEHI 231 cell survival and implicated activation of the anti-apoptotic factors Survivin and manganese superoxide dismutase. Thus, CD40 engagement of transformed B cells activates relB gene transcription via a process we have termed the de novo RelB synthesis pathway, which protects these cells from apoptosis.


Assuntos
Apoptose/fisiologia , Linfócitos B/fisiologia , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição RelB/metabolismo , Animais , Linfócitos B/citologia , Antígenos CD40/genética , Ligante de CD40/genética , Linhagem Celular Tumoral , Antígeno 2 Relacionado a Fos/genética , Antígeno 2 Relacionado a Fos/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Leucemia Linfocítica Crônica de Células B/imunologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Survivina , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição RelB/genética , Transcrição Gênica
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