RESUMEN
MYC is a transcriptional factor that regulates growth and proliferation through cell cycle pathways. MYC alterations, in particular MYC rearrangements, are important in assessing the prognosis of aggressive B-cell lymphoma. In this study, we focused on the impact of nine major cell cycle genes for MYC-driven aggressive mature B-cell lymphoma and analyzed the mutational status using targeted next generation sequencing. Our 40 cases of aggressive mature B-cell lymphomas included 5 Burkitt lymphomas, 17 high-grade B-cell lymphomas and 18 diffuse large B-cell lymphomas with MYC breaks in 100%, 88% and 11%, respectively. Our data allowed a molecular classification into four categories partially independent from the histopathological diagnosis but correlating with the Ki-67 labelling index: (I) harboring TP53 and CDKN2A mutations, being highly proliferative, (II) with MYC rearrangement associated with MYC and/or ID3 mutations, being highly proliferative, (III) with MYC rearrangement combined with additional molecular changes, being highly proliferative, and (IV) with a diverse pattern of molecular alterations, being less proliferative. Taken together, we found that mutations of TP53, CDKN2A, MYC and ID3 are associated with highly proliferative B-cell lymphomas that could profit from novel therapeutic strategies.
Asunto(s)
Ciclo Celular , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico , Humanos , Linfoma de Células B/diagnóstico , Linfoma de Células B/patología , Mutación , PronósticoRESUMEN
PURPOSE: To analyze the impact of TNFα or IL2 on human lymphocytes in vitro and the anti-tumor and immune-modifying effects of L19-IL2 and L19-TNFα on subcutaneously growing J558L myeloma in immunocompetent mice. METHODS: PBMCs from three healthy volunteers were incubated with IL2, TNFα, or with IL2 plus addition of TNFα (final 20 h). BALB/c J558L mice with subcutaneous tumors were treated with intravenous L19-TNFα plus L19-IL2, or controls. Tumor growth and intra- and peri-tumoral tissues were analyzed for micro-vessel density, necrosis, immune cell composition, and PD1 or PD-L1 expressing cells. RESULTS: Exposure of PBMC in vitro to IL2, TNFα, or to IL2 over 3 and 5 days plus TNFα for the final 20 h resulted in an approximately 50 and 75% reduction of the CD25low effector cell/CD25high Treg cell ratio, respectively, compared to medium control. IL2 or TNFα increased the proportion of CD4- CD25low effector lymphocytes while reducing the proportion of CD4+ CD25low Teff cells. In the J558L myeloma model, tumor eradication was observed in 58, 42, 25, and 0% of mice treated with L19-TNFα plus L19-IL2, L19-TNFα, L19-IL2, and PBS, respectively. L19-TNFα/L19-IL2 combination caused tumor necrosis, capillary density doubling, peri-tumoral T cell and PD1+ T cell reduction (- 50%), and an increase in PD-L1+ myeloma cells. CONCLUSION: IL2, TNFα, or IL2 plus TNFα (final 20 h) increased the proportion of CD4- CD25low effector lymphocytes possibly indicating immune activation. L19-TNFα/L19-IL2 combination therapy eradicated tumors in J558L myeloma BALB/c mice likely via TNFα-induced tumor necrosis and L19-TNFα/L19-IL2-mediated local cellular immune reactions.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Inmunoterapia/métodos , Interleucina-2/uso terapéutico , Mieloma Múltiple/terapia , Neovascularización Patológica/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/uso terapéutico , Animales , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Inmunotoxinas/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Mieloma Múltiple/inmunología , Mieloma Múltiple/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Trasplante IsogénicoRESUMEN
Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are not commonly used in clinical practice for treatment of B-cell lymphomas, although a subset of patients with refractory or relapsed B-cell lymphoma achieved partial or complete remissions. Therefore, the purpose of this study was to identify molecular features that predict the response of B-cell lymphomas to SAHA treatment. We designed an integrative approach combining drug efficacy testing with exome and captured target analysis (DETECT). In this study, we tested SAHA sensitivity in 26 B-cell lymphoma cell lines and determined SAHA-interacting proteins in SAHA resistant and sensitive cell lines employing a SAHA capture compound (CC) and mass spectrometry (CCMS). In addition, we performed exome mutation analysis. Candidate validation was done by expression analysis and knock-out experiments. An integrated network analysis revealed that the Src tyrosine kinase Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog (FGR) is associated with SAHA resistance. FGR was specifically captured by the SAHA-CC in resistant cells. In line with this observation, we found that FGR expression was significantly higher in SAHA resistant cell lines. As functional proof, CRISPR/Cas9 mediated FGR knock-out in resistant cells increased SAHA sensitivity. In silico analysis of B-cell lymphoma samples (n = 1200) showed a wide range of FGR expression indicating that FGR expression might help to stratify patients, which clinically benefit from SAHA therapy. In conclusion, our comprehensive analysis of SAHA-interacting proteins highlights FGR as a factor involved in SAHA resistance in B-cell lymphoma.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Linfoma de Células B/patología , Proteínas Proto-Oncogénicas/metabolismo , Familia-src Quinasas/metabolismo , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Espectrometría de Masas , Mutación/genética , Reproducibilidad de los Resultados , VorinostatRESUMEN
BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. The typical and atypical carcinoid (TC and AC), the large-cell neuroendocrine carcinoma (LCNEC) and the small-cell lung cancers (SCLC) are subgroups of pulmonary tumours that show neuroendocrine differentiations. With the rising impact of molecular pathology in routine diagnostics the interest for reliable biomarkers, which can help to differentiate these subgroups and may enable a more personalised treatment of patients, grows. METHODS: A collective of 70 formalin-fixed, paraffin-embedded (FFPE) pulmonary neuroendocrine tumours (17 TCs, 17 ACs, 19 LCNECs and 17 SCLCs) was used to identify biomarkers by high-throughput sequencing. Using the Illumina TruSeq Amplicon-Cancer Panel on the MiSeq instrument, the samples were screened for alterations in 221 mutation hot spots of 48 tumour-relevant genes. RESULTS: After filtering >26 000 detected variants by applying strict algorithms, a total of 130 mutations were found in 29 genes and 49 patients. Mutations in JAK3, NRAS, RB1 and VHL1 were exclusively found in SCLCs, whereas the FGFR2 mutation was detected in LCNEC only. KIT, PTEN, HNF1A and SMO were altered in ACs. The SMAD4 mutation corresponded to the TC subtype. We prove that the frequency of mutations increased with the malignancy of tumour type. Interestingly, four out of five ATM-mutated patients showed an additional alteration in TP53, which was by far the most frequently altered gene (28 out of 130; 22%). We found correlations between tumour type and IASLC grade for ATM- (P=0.022; P=0.008) and TP53-mutated patients (P<0.001). Both mutated genes were also associated with lymph node invasion and distant metastasis (P⩽0.005). Furthermore, PIK3CA-mutated patients with high-grade tumours showed a reduced overall survival (P=0.040) and the mutation frequency of APC and ATM in high-grade neuroendocrine lung cancer patients was associated with progression-free survival (PFS) (P=0.020). CONCLUSIONS: The implementation of high-throughput sequencing for the analysis of the neuroendocrine lung tumours has revealed that, even if these tumours encompass several subtypes with varying clinical aggressiveness, they share a number of molecular features. An improved understanding of the biology of neuroendocrine tumours will offer the opportunity for novel approaches in clinical management, resulting in a better prognosis and prediction of therapeutic response.
Asunto(s)
Neoplasias Pulmonares/genética , Mutación , Tumores Neuroendocrinos/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Tumores Neuroendocrinos/patología , Adhesión en Parafina , Adulto JovenRESUMEN
PURPOSE: The extradomain B of fibronectin (ED-B) is a promising vascular target for selective pharmacodelivery in cancer patients. We analyzed a large series of prostatectomies from patients with prostate cancer, hyperplastic prostate disease, and normal prostates to study extent and tumor-selectivity of ED-B expression. METHODS: Using immunohistology, 68 adenocarcinomas of the prostate or prostate cancer-inflicted lymph nodes, 4 samples of benign prostatic hyperplasia, and 6 normal prostate glands were studied for ED-B expressing newly formed blood vessels. Further, we treated an advanced prostate cancer patient with the anti-ED-B antibody (131)I-L19SIP to study in vivo target accessibility. RESULTS: ED-B-positive blood vessels were found significantly more frequent in prostate cancers as compared with peritumoral prostate tissues or normal prostate glands, independent of tumor differentiation. The ED-B-positive blood vessels' density was 97 (±23), 65 (±9), and 59 (±9)/mm(2) in G3, G2, and G1 prostate cancers, respectively, and 7 (±5)/mm(2) in normal prostate glands. In high-grade (G3) prostate cancers, also the peritumoral tissue showed a higher density of ED-B vessels than normal prostate glands. Similar results were obtained when ED-B-positive vessel density was expressed as a fraction of CD34-positive vessel density. Finally, selective uptake of ED-B-binding (131)I-L19SIP to tumor lesions was found in an advanced prostate cancer patient by whole-body planar scintigraphy. CONCLUSIONS: ED-B-positive blood vessels were found to a large extent in prostate cancer tissues, but only rarely in normal prostates or benign prostatic hyperplasia. Whole-body planar scintigraphy in a prostate cancer patient confirmed selective uptake of (131)I-L19SIP in the prostate cancer tissues, qualifying ED-B as a promising target for selective pharmacodelivery of anticancer agents in prostate cancer.
Asunto(s)
Fibronectinas/biosíntesis , Inmunotoxinas/farmacocinética , Radioisótopos de Yodo/farmacocinética , Neoplasias de la Próstata/metabolismo , Radiofármacos/farmacocinética , Proteínas Recombinantes de Fusión/farmacocinética , Anticuerpos/inmunología , Vasos Sanguíneos/metabolismo , Epítopos/análisis , Humanos , Inmunohistoquímica , Masculino , Microscopía Confocal , Persona de Mediana Edad , Neovascularización Patológica/diagnóstico por imagen , Neovascularización Patológica/metabolismo , Adhesión en Parafina , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Neoplasias de la Próstata/irrigación sanguínea , Neoplasias de la Próstata/diagnóstico por imagen , Neoplasias de la Próstata/patología , Radioinmunodetección , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, is consistently expressed by tumor cells of anaplastic large-cell lymphoma (ALCL). CD30 stimulation induces massive caspase-dependent cell death of ALCL cells in case of canonical NFκB inhibition or proteasome inhibition. However, CD30, a TNFR lacking a death domain (DD), is unable to recruit a death inducing complex containing TRADD (TNFR1-associated DD-protein) or FADD (FAS-associated DD-domain protein) together with the receptor-interacting protein 1 (RIP1) and caspase-8. Thus, the mechanism explaining CD30-induced cell death of lymphocytes remains obscure. Here, we demonstrate that blockage of RIP1 by siRNA or pharmacological inhibition of RIP1 by Necrostatin-1 almost completely prevented CD30-induced cell death. In addition, we revealed CD30-induced accumulation of RIP1 at the cytoplasma membrane of NFκB-inhibited ALCL cells by confocal laser scanning microscopy. Finally, primary ALCL cases can be subdivided into two groups based on the presence or absence of RIP1 as revealed by immunohistology. Taken together, our study identified RIP1 as a crucial mediator of CD30-induced cell death that bears features of apoptosis as well as necroptosis. RIP1 expression in ALCL tumor cells might eligible for the therapeutic application of CD30 antibodies in combination with NFκB/proteasome inhibitors that should result in CD30-induced cell death.
Asunto(s)
Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Comunicación Autocrina , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Benzoquinonas , Muerte Celular , Membrana Celular/metabolismo , Células HeLa , Humanos , Imidazoles , Indoles , Proteínas Inhibidoras de la Apoptosis/metabolismo , Lactamas Macrocíclicas , FN-kappa B/metabolismo , Factor 1 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
AIMS: A20 (TNFAIP3) is a nuclear factor-κB (NF-κB)-inducible component of tumour necrosis factor and Toll-like receptor intracellular signal transduction. It negatively regulates NF-κB, and has been identified as a tumour suppressor. Several studies have described A20 inactivation by deletion of the A20 locus at 6q23, inactivating mutations, and/or methylation of the A20 promoter in various lymphoma entities. METHODS AND RESULTS: We generated a monoclonal antibody against the C-terminus of A20 (Ber-A20) and investigated full-length A20 expression of normal lymphoid tissue and lymphomas for the first time. We identified loss of A20 expression in tumour cells of 24% of classical Hodgkin lymphoma, 27% of diffuse large B-cell lymphoma, 20% of chronic lymphocytic leukaemia, 19% of follicular lymphoma, 13% of mantle cell lymphoma and 8% of primary mediastinal B-cell lymphoma cases by immunohistology. Loss of A20 expression rarely occurred in T-cell non-Hodgkin lymphoma. CONCLUSIONS: Our data are in agreement with cytogenetic and molecular analyses. Among 21 cases of ocular adnexal marginal zone lymphomas with known A20 mutation status, we detected complete absence of A20 expression, whereas cases with wild-type A20 were weakly A20-positive. We demonstrate that A20 loss can be detected by immunohistology with a sensitivity similar to that of complex molecular and genetic methods.
Asunto(s)
Anticuerpos Monoclonales/química , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Anticuerpos Monoclonales/metabolismo , Proteínas de Unión al ADN/inmunología , Neoplasias del Ojo/metabolismo , Enfermedad de Hodgkin/metabolismo , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/inmunología , Linfoma no Hodgkin/metabolismo , Mutación , Proteínas Nucleares/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
Endogenous ß-galactose-binding lectins have many biological functions, but their biological significance in Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) remains unclear. By immunohistochemistry, we analyzed the expression of galectin-1 and galectin-3 in HL and ALCL cases as well as in cell lines, and investigated the pharmacological effects of galectin-1 treatment with and without CD30 pre-stimulation of HL and ALCL cell lines. The galectin-3-negative human embryonic kidney cell line (HEK-293) was transfected with galectin-3 cDNA. Galectin-3 is differentially expressed in HL and ALCL. CD30 stimulation of the ALCL cell line Karpas 299 activates NF-κB without induction of apoptosis. Galectin-1 treatment of Karpas 299 induces cell death, which is significantly increased by CD30 pre-stimulation. The CD30-mediated increase of galectin-1-induced cell death is to some extent caspase independent and does not influence the expression of tumor necrosis factor-associated factor 1 (TRAF1), TRAF2, and cellular inhibitor of apoptosis 2 protein (cIAP2), as revealed in Karpas 299 cells. In other cell lines except Karpas 299, CD30 pre-stimulation did not significantly enhance galectin-1-induced cell death. Galectin-3 transfection of HEK-293 cells resulted in cell surface expression of galectin-3, associated with marked cell aggregation. CD30-targeted therapy in combination with galectin-1 treatment may induce effective killing of ALCL cells but not of HL cells.
Asunto(s)
Apoptosis/fisiología , Galectina 1/fisiología , Enfermedad de Hodgkin/patología , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/patología , Transducción de Señal/fisiología , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Cartilla de ADN , Citometría de Flujo , Enfermedad de Hodgkin/metabolismo , Humanos , Inmunohistoquímica , Linfoma Anaplásico de Células Grandes/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has been conducted in BL so far. METHODOLOGY/PRINCIPAL FINDINGS: ChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing. CONCLUSION/SIGNIFICANCE: The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology.
Asunto(s)
Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , ADN de Neoplasias/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Sitios de Unión , Biomarcadores de Tumor/genética , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Células Tumorales CultivadasRESUMEN
Death ligands, including TNF-alpha, CD95L/FasL, and TRAIL, mediate safeguard mechanisms against tumor growth and critically contribute to lymphocyte homeostasis. We investigated death receptor-mediated apoptosis and CD30/CD95 crosstalk in four CD30-positive cell lines of cutaneous anaplastic large-cell lymphoma (cALCL). Whereas CD95 stimulation strongly induced apoptosis in cALCL cells, the pro-apoptotic pathways of TNF-alpha and TRAIL were completely blocked at an early step. Expression of TNF receptor 1 was lost in three of four cell lines, providing an explanation for TNF-alpha unresponsiveness. TRAIL resistance may be explained by the consistent overexpression of cellular flice inhibitory protein (c-FLIP) (four of four cell lines) and frequent loss of the pro-apoptotic Bcl-2 protein Bid (three of four cell lines). Changes at the receptor-expression level were largely ruled out. CD30/CD95 crosstalk experiments showed that CD30 ligation leads to NF-kappaB-mediated c-FLIP upregulation in cALCL cells, which in turn conferred enhanced resistance to CD95-mediated apoptosis. Knockdown of c-FLIP by a lentiviral approach enhanced basic apoptosis rates in cALCL cells and diminished the CD30-mediated suppression of apoptosis, thus proving the significance of c-FLIP in this context. These in vitro findings may be indicative of the clinical situation of cALCL. Further clarifying the defects in apoptosis pathways in cutaneous lymphomas may lead to improved therapies for these disorders.
Asunto(s)
Apoptosis/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/patología , Neoplasias Cutáneas/patología , Apoptosis/efectos de los fármacos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Antígeno Ki-1/farmacología , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/fisiopatología , FN-kappa B/metabolismo , Interferencia de ARN , Receptor Cross-Talk/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/fisiopatología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Receptor fas/metabolismoRESUMEN
Current treatment of hematologic malignancies involves rather unspecific chemotherapy, frequently resulting in severe adverse events. Thus, modern clinical research focuses on compounds able to discriminate malignant from normal tissues. Being expressed in newly formed blood vessels of solid cancers but not in normal mature tissues, the extradomain B of fibronectin (ED-B FN) is a promising target for selective cancer therapies. Using immunohistology with a new epitope retrieval technique for paraffin-embedded tissues, ED-B FN expression was found in biopsies from more than 200 Hodgkin and non-Hodgkin lymphoma patients of nearly all entities, and in patients with myeloproliferative diseases. ED-B FN expression was nearly absent in normal lymph nodes (n = 10) and bone marrow biopsies (n = 9). The extent of vascular ED-B FN expression in lymphoma tissues was positively correlated with grade of malignancy. ED-B FN expression was enhanced in lymph nodes with severe lymphadenopathy and in some hyperplastic tonsils. The in vivo accessibility of ED-B FN was confirmed in 3 lymphoma patients, in whom the lymphoma lesions were visualized on scintigraphy with (131)I-labeled L19 small immunoprotein ((131)I-L19SIP). In 2 relapsed Hodgkin lymphoma patients(131)I-L19SIP radioimmunotherapy induced a sustained partial response, qualifying ED-B FN as a promising target for antibody-based lymphoma therapies.
Asunto(s)
Anticuerpos/uso terapéutico , Fibronectinas/biosíntesis , Enfermedad de Hodgkin/radioterapia , Radioinmunoterapia/métodos , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Técnica del Anticuerpo Fluorescente , Glucosa-6-Fosfato/análogos & derivados , Enfermedad de Hodgkin/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Tomografía de Emisión de Positrones , Isoformas de Proteínas/biosíntesis , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
High CD30 expression in classical Hodgkin's lymphoma and anaplastic large cell lymphoma (ALCL) suggests an important pathogenic role of this cytokine receptor. To test this hypothesis, we investigated CD30 signaling in Hodgkin's and ALCL cell lines by different approaches: 1) CD30 stimulation, 2) CD30 down-regulation, and 3) a combination of both. The effects were determined at the RNA (microarray and real-time quantitative RT-PCR), protein (electrophoretic mobility shift analysis, immunoblot, and flow cytometry), and cellular/functional (proliferation and apoptosis) levels. We demonstrate that Hodgkin's cells are virtually CD30 unresponsive. Neither CD30 stimulation nor CD30 silencing of Hodgkin's cells had any significant effect. In contrast, CD30 stimulation of ALCL cells activated nuclear transcription factor-kappaB (NF-kappaB), induced major transcriptional changes, and decreased proliferation. These effects could be abrogated by down-regulation of CD30. Stimulation of CD30 in ALCL cells, stably transfected with a dominant-negative NF-kappaB inhibitor, induced pronounced caspase activation and massive apoptosis. Our data indicate that 1) CD30 signaling is not effective in Hodgkin's cell lines but is effective in ALCL cell lines, 2) CD30 is probably not significantly involved in the pathogenesis of classical Hodgkin's lymphoma, and 3) CD30 stimulation triggers two competing effects in ALCL cells, namely activation of caspases and NF-kappaB-mediated survival. These data suggest that CD30-targeted therapy in ALCL should be combined with NF-kappaB inhibitors to induce effective cell killing.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/metabolismo , Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/metabolismo , Transducción de Señal/fisiología , Apoptosis/fisiología , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática/fisiología , Citometría de Flujo , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Immunoblotting , FN-kappa B/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Although the therapy of Hodgkin lymphoma and anaplastic large cell lymphoma has been considerably improved during the last decades, high therapeutic toxicity, relapses, secondary tumors, and primary treatment failure(s) occur. Both malignancies are well suited for CD30-targeted immunotherapy because of their strong CD30 expression. We constructed an immunotoxin composed of a single chain variable fragment of a CD30 antibody fused to the human pancreatic ribonuclease, showing CD30-specific binding and ribonucleolytic activity resistant to the inhibitor RNasin. This immunotoxin revealed CD30-specific anti-tumor activity in BALB/c mice that were challenged with CD30-positive or CD30-negative syngeneic tumor cells.
Asunto(s)
Citotoxinas/farmacología , Inmunotoxinas/farmacología , Antígeno Ki-1/inmunología , Ribonucleasa Pancreática/inmunología , Animales , Especificidad de Anticuerpos , Línea Celular , Proliferación Celular/efectos de los fármacos , Drosophila melanogaster , Femenino , Humanos , Antígeno Ki-1/química , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias/patología , Unión Proteica , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleasa Pancreática/química , Trasplante Isogénico/patología , Células Tumorales CultivadasRESUMEN
Lymphomatoid papulosis (LyP), primary cutaneous anaplastic large T-cell lymphoma (cALCL), and cutaneous infiltrates of systemic anaplastic large cell lymphoma (sALCL) are CD30-positive lymphoproliferative disorders of the skin that overlap clinically, histopathologically, immunophenotypically, and genetically but differ considerably in their prognosis. In particular, lesions of LyP regress spontaneously, whereas those of cALCL and sALCL persist and may progress and spread to extracutaneous sites. In contrast to patients with cALCL, LyP patients do not benefit from an aggressive radio- and/or chemotherapeutic approach. We generated a novel tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) antibody that recognizes a formalin-resistant epitope (Ber-TRAF1A) and investigated the expression of TRAF1, an intracellular component of TNFR signaling, in LyP and ALCL. We could show a strong TRAF1 expression in the tumor cells of most LyP cases (42/49, 84%). In contrast, tumor cells of primary and secondary cALCL revealed TRAF1 expression in only a few cases (3/41, 7%) as shown for sALCL without skin manifestation. The data indicate that TRAF1 expression reliably distinguishes LyP from primary or secondary cALCL. This might be of crucial diagnostic importance and has a strong impact on the treatment decision for patients with cALCL and LyP.
Asunto(s)
Antígeno Ki-1/análisis , Linfoma de Células B Grandes Difuso/química , Linfoma Cutáneo de Células T/química , Linfoma de Células T/química , Papulosis Linfomatoide/metabolismo , Factor 1 Asociado a Receptor de TNF/análisis , Animales , Humanos , Ratones , Ratones Endogámicos BALB C , Factor 1 Asociado a Receptor de TNF/inmunología , Factor 1 Asociado a Receptor de TNF/fisiologíaRESUMEN
In this study, the expression of activated caspase-3 by the tumor cells of classical Hodgkin lymphoma (cHL), the Hodgkin-Reed-Sternberg (HRS) cells, is confirmed. This raises the question why caspase-3 does not kill HRS cells. There are only a few molecules, which are able to directly inhibit caspase-3. One of them is cIAP2. We show that cIAP2 is expressed in the HRS cells in 20 of 23 cHL cases by in situ hybridization. Suppression experiments with cIAP2 antisense RNA show that down-regulation of cIAP2 significantly reduces apoptosis resistance in cHL cell lines. cIAP2 overexpression appears to be unique for HRS cells since the tumor cells of non-Hodgkin lymphomas are nearly cIAP2-negative. We demonstrate that cIAP2 is inducible by CD30 stimulation in cHL cell lines of T-cell origin and anaplastic large cell lymphoma cell lines, whereas cHL cell lines of B-cell origin constitutively express cIAP2. Inhibition of cIAP2 expression by cIAP2 antisense RNA decreases resistance to apoptosis. The results indicate that cIAP2 contributes to the apoptosis resistance of HRS cells, mainly by inhibiting effector caspases. According to these findings, a therapeutical application of inhibitors of apoptosis proteins antagonists in cHL appears promising.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Células de Reed-Sternberg/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Células HeLa , Humanos , Hibridación in Situ , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/genética , ARN sin Sentido/farmacología , Células de Reed-Sternberg/patologíaRESUMEN
Resistance to death receptor-mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain-containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1beta-converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-kappaB. Despite expression of other NF-kappaB-dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/fisiología , Proteínas Portadoras/fisiología , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/fisiopatología , Péptidos y Proteínas de Señalización Intracelular , Células de Reed-Sternberg/patología , Células de Reed-Sternberg/fisiología , Receptor fas/fisiología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasa 10 , Caspasa 8 , Caspasas/metabolismo , Línea Celular Tumoral , Cicloheximida/farmacología , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Glicoproteínas de Membrana/fisiología , FN-kappa B/metabolismo , ARN Interferente Pequeño/genética , Células de Reed-Sternberg/efectos de los fármacos , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
A20 and TRAF1 are two anti-apoptotic components of the intracellular signalling pathway of the tumour necrosis factor receptor (TNFR) family. Induction of apoptosis seems to be a main function of these receptors. It is astonishing that a member of this family, CD30, is overexpressed by highly proliferating tumours such as Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). It is known that CD30 stimulation leads to the apoptosis of ALCL tumour cells but not of Hodgkin-Reed-Sternberg (HRS) cells. We have already established the overexpression of TRAF1 in HRS cells. In this study we demonstrate that A20 is highly expressed in the HRS cells in 20/22 of cases of classical HL, in 4/4 cases of nodular lymphocyte-predominant HL (NLPHL), and in 2/2 cases of the anaplastic variant of diffuse large B cell lymphoma. In contrast, all other non-Hodgkin lymphomas, including ALCL, revealed either no A20 reactivity, or reactivity in less than 1% of all tumour cells. CD30 stimulation induced A20 and TRAF1 expression. This effect was most prominent in HL and ALCL cell lines with low basal expression levels of these molecules. Immunohistological studies of reactive lymphoid blasts in tonsillar tissue demonstrated that co-expression of CD30, A20, and TRAF1 also occurs in vivo. Cell lines with high basal A20 and TRAF1 expression were resistant to CD30-mediated apoptosis. The sensitivity to CD30-induced apoptosis was increased by inhibition of protein synthesis. TRAF1 transfection decreased CD30-induced apoptosis. Our data suggest that A20 and TRAF1 contribute to apoptosis resistance and, therefore, play an important role in the pathogenesis of classical HL.