RESUMO
Arginine methyltransferases critically regulate cellular homeostasis by modulating the functional outcome of their substrates. The protein arginine methyltransferase 5 (PRMT5) is an enzyme involved in growth and survival pathways promoting tumorigenesis. However, little is known about the biologic function of PRMT5 and its therapeutic potential in multiple myeloma (MM). In the present study, we identified and validated PRMT5 as a new therapeutic target in MM. PRMT5 is overexpressed in patient MM cells and associated with decreased progression-free survival and overall survival. Either genetic knockdown or pharmacological inhibition of PRMT5 with the inhibitor EPZ015666 significantly inhibited growth of both cell lines and patient MM cells. Furthermore, PRMT5 inhibition abrogated NF-κB signaling. Interestingly, mass spectrometry identified a tripartite motif-containing protein 21 TRIM21 as a new PRMT5-partner, and we delineated a TRIM21-dependent mechanism of NF-κB inhibition. Importantly, oral administration of EPZ015666 significantly decreased MM growth in a humanized murine model of MM. These data both demonstrate the oncogenic role and prognostic relevance of PRMT5 in MM pathogenesis, and provide the rationale for novel therapies targeting PRMT5 to improve patient outcome.
Assuntos
Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Humanos , Isoquinolinas/farmacologia , NF-kappa B/metabolismo , Prognóstico , Pirimidinas/farmacologia , Ribonucleoproteínas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.
Assuntos
Reparo do DNA/genética , Mieloma Múltiplo/genética , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Humanos , Transcrição Gênica/genética , Xeroderma Pigmentoso/genéticaRESUMO
Epigenetic signaling pathways are implicated in tumorigenesis and therefore histone deacetylases (HDACs) represent novel therapeutic targets for cancers, including multiple myeloma (MM). Although non-selective HDAC inhibitors show anti-MM activities, unfavorable side effects limit their clinical efficacy. Isoform- and/or class-selective HDAC inhibition offers the possibility to maintain clinical activity while avoiding adverse events attendant to broad non-selective HDAC inhibition. We have previously reported that HDAC3 inhibition, either by genetic knockdown or selective inhibitor BG45, abrogates MM cell proliferation. Here we show that knockdown of HDAC3, but not HDAC1 or HDAC2, as well as BG45, downregulate expression of DNA methyltransferase 1 (DNMT1) mediating MM cell proliferation. DNMT1 expression is regulated by c-Myc, and HDAC3 inhibition triggers degradation of c-Myc protein. Moreover, HDAC3 inhibition results in hyperacetylation of DNMT1, thereby reducing the stability of DNMT1 protein. Combined inhibition of HDAC3 and DNMT1 with BG45 and DNMT1 inhibitor 5-azacytidine (AZA), respectively, triggers synergistic downregulation of DNMT1, growth inhibition and apoptosis in both MM cell lines and patient MM cells. Efficacy of this combination treatment is confirmed in a murine xenograft MM model. Our results therefore provide the rationale for combination treatment using HDAC3 inhibitor with DNMT1 inhibitor to improve patient outcome in MM.
Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Acetilação , Animais , Apoptose , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Modelos Biológicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Interferência de RNA , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent studies have delineated cancer-type-specific roles of histone 3 lysine 27 (H3K27) demethylase KDM6B/JMJD3 depending on its H3K27 demethylase activity. Here we show that KDM6B is expressed in multiple myeloma (MM) cells; and that shRNA-mediated knockdown and CRISPR-mediated knockout of KDM6B abrogate MM cell growth and survival. Tumor necrosis factor-α or bone marrow stromal cell culture supernatants induce KDM6B, which is blocked by IKKß inhibitor MLN120B, suggesting that KDM6B is regulated by NF-κB signaling in MM cells. RNA-seq and subsequent ChIP-qPCR analyses reveal that KDM6B is recruited to the loci of genes encoding components of MAPK signaling pathway including ELK1 and FOS, and upregulates expression of these genes without affecting H3K27 methylation level. Overexpression of catalytically inactive KDM6B activates expression of MAPK pathway-related genes, confirming its function independent of demethylase activity. We further demonstrate that downstream targets of KDM6B, ELK1 and FOS, confer MM cell growth. Our study therefore delineates KDM6B function that links NF-κB and MAPK signaling pathway mediating MM cell growth and survival, and validates KDM6B as a novel therapeutic target in MM.
Assuntos
Histona Desmetilases com o Domínio Jumonji/metabolismo , Sistema de Sinalização das MAP Quinases , Mieloma Múltiplo/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
This corrects the article DOI: 10.1038/leu.2016.388.
RESUMO
Proteasome inhibition is an effective therapy for multiple myeloma (MM) patients; however, the emergence of drug resistance is common. Novel therapeutic strategies to overcome proteasome inhibitor resistance are needed. In this study, we examined whether targeting deubiquitylating (DUB) enzymes upstream of 20S proteasome overcomes proteasome inhibitor resistance. Gene expression analysis, immunohistochemical studies of MM patient bone marrow, reverse transcription-PCR and protein analysis show that Rpn11/POH1, a DUB enzyme upstream of 20S proteasome, is more highly expressed in patient MM cells than in normal plasma cells. Importantly, Rpn11 expression directly correlates with poor patient survival. Loss-of-function studies show that Rpn11-siRNA knockdown decreases MM cell viability. Pharmacological inhibition of Rpn11 with O-phenanthroline (OPA) blocks cellular proteasome function, induces apoptosis in MM cells and overcomes resistance to proteasome inhibitor bortezomib. Mechanistically, Rpn11 inhibition in MM cells activates caspase cascade and endoplasmic stress response signaling. Human MM xenograft model studies demonstrate that OPA treatment reduces progression of tumor growth and prolongs survival in mice. Finally, blockade of Rpn11 increases the cytotoxic activity of anti-MM agents lenalidomide, pomalidomide or dexamethasone. Overall, our preclinical data provide the rationale for targeting DUB enzyme Rpn11 upstream of 20S proteasome to enhance cytotoxicity and overcome proteasome inhibitor resistance in MM.
Assuntos
Antineoplásicos/uso terapêutico , Bortezomib/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/uso terapêutico , Transativadores/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Mieloma Múltiplo/enzimologia , Fenantrolinas/farmacologia , Prognóstico , Complexo de Endopeptidases do Proteassoma , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3É (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antígeno de Maturação de Linfócitos B/imunologia , Complexo CD3/imunologia , Mieloma Múltiplo/terapia , Linfócitos T/imunologia , Animais , Apoptose , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Ativação Linfocitária , Macaca fascicularis , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The BAX proapoptotic protein is capable of inducing cell death either directly, through its effects on mitochondrial function, or indirectly, by lowering the apoptotic threshold in response to certain chemotherapy agents. In this study, we tested the hypothesis that selective expression of BAX in human ovarian cancer through adenoviral gene transfer might represent a novel approach to eradicating tumor cells in vivo. Two constructs were prepared using replication-deficient adenoviral vectors containing either the cDNA for beta-galactosidase (Ad.DF3.betaGAL) or hemagglutinin (HA)-tagged BAX (Ad.DF3.BAX) under the control of the DF3 promoter. The DF3 promoter was used to confer tumor-specific gene expression in view of its restricted pattern of expression in the majority of human ovarian cancers and its limited expression in normal peritoneal mesothelial cells. In vitro infection of up to seven different epithelial cancer cell lines with Ad.DF3.betaGAL or Ad.DF3.BAX resulted in expression of either beta-galactosidase activity or HA-BAX protein, respectively, which was highly correlated with DF3 levels. Furthermore, infection with Ad.DF3.BAX was capable of highly selective cytotoxicity of DF3-positive ovarian cancer clonogenic cells in vitro. The effect of i.p. administration of Ad.DF3.BAX was also assessed in nude mice inoculated with the DF3-positive 36M2 human ovarian cancer cell line. Expression of either beta-galactosidase activity (after Ad.DF3.betaGAL treatment) or HA-BAX transcripts (after Ad.DF3.BAX treatment) was restricted to tumor tissue in vivo. Importantly, administration of Ad.DF3.BAX on days 2 and 3 after tumor inoculation was capable of eradicating >99% of tumor implants. These results demonstrate the feasibility of tumor selective expression of a proapoptotic protein such as BAX through adenoviral gene transfer.
Assuntos
Antígenos de Neoplasias/genética , Apoptose/genética , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas/fisiologia , Adenocarcinoma/patologia , Adenovírus Humanos/genética , Animais , Neoplasias da Mama/patologia , Estudos de Viabilidade , Feminino , Genes Reporter , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Injeções Intraperitoneais , Óperon Lac , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Regiões Promotoras Genéticas , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Células Tumorais Cultivadas/metabolismo , Proteína X Associada a bcl-2 , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
Previous studies have demonstrated that gamma-irradiation (IR)-induced apoptosis in multiple myeloma (MM) is associated with activation of stress-activated protein kinase (SAPK). In the present study, we examined the molecules downstream of SAPK/C-Jun N-terminal kinase (JNK), focusing on the role of retinoblastoma protein (Rb) during IR-induced MM cell apoptosis. The results demonstrate that IR activates SAPK/JNK, which associates with Rb both in vivo and in vitro. Far Western blot analysis confirms that SAPK/JNK binds directly to Rb. IR-activated SAPK/JNK phosphorylates Rb, and deletion of the phosphorylation site in the COOH terminus domain of Rb abrogates phosphorylation of Rb by SAPK/JNK. Taken together, our results suggest that Rb is a target protein of SAPK/JNK and that the association of SAPK/JNK and Rb mediates IR-induced apoptosis in MM cells.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Ativadas por Mitógeno , Mieloma Múltiplo/metabolismo , Proteína do Retinoblastoma/metabolismo , Apoptose/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Raios gama , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Mieloma Múltiplo/enzimologia , Fosforilação/efeitos da radiação , Ligação Proteica , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
Proteasome inhibitor bortezomib is an effective therapy for relapsed and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance can limit its long-term utility. Recent research has focused on targeting ubiquitin receptors upstream of 20S proteasome, with the aim of generating less toxic therapies. Here we show that 19S proteasome-associated ubiquitin receptor Rpn13 is more highly expressed in MM cells than in normal plasma cells. Rpn13-siRNA (small interfering RNA) decreases MM cell viability. A novel agent RA190 targets Rpn13 and inhibits proteasome function, without blocking the proteasome activity or the 19S deubiquitylating activity. CRISPR/Cas9 Rpn13-knockout demonstrates that RA190-induced activity is dependent on Rpn13. RA190 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma and overcomes bortezomib resistance. Anti-MM activity of RA190 is associated with induction of caspase-dependent apoptosis and unfolded protein response-related apoptosis. MM xenograft model studies show that RA190 is well tolerated, inhibits tumor growth and prolongs survival. Combining RA190 with bortezomib, lenalidomide or pomalidomide induces synergistic anti-MM activity. Our preclinical data validates targeting Rpn13 to overcome bortezomib resistance, and provides the framework for clinical evaluation of Rpn13 inhibitors, alone or in combination, to improve patient outcome in MM.
Assuntos
Compostos de Benzilideno/uso terapêutico , Glicoproteínas de Membrana/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Animais , Apoptose , Compostos de Benzilideno/farmacologia , Bortezomib/uso terapêutico , Proliferação de Células , Sobrevivência Celular , Sinergismo Farmacológico , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Terapia de Alvo Molecular , Células Tumorais CultivadasRESUMO
The anti-CD38 monoclonal antibody SAR650984 (SAR) is showing promising clinical activity in treatment of relapsed and refractory multiple myeloma (MM). Besides effector-mediated antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity, we here define molecular mechanisms of SAR-directed MM cell death and enhanced anti-MM activity triggered by SAR with Pomalidomide (Pom). Without Fc-cross-linking agents or effector cells, SAR specifically induces homotypic aggregation (HA)-associated cell death in MM cells dependent on the level of cell surface CD38 expression, actin cytoskeleton and membrane lipid raft. SAR and its F(ab)'2 fragments trigger caspase 3/7-dependent apoptosis in MM cells highly expressing CD38, even with p53 mutation. Importantly, SAR specifically induces lysosome-dependent cell death (LCD) by enlarging lysosomes and increasing lysosomal membrane permeabilization associated with leakage of cathepsin B and LAMP-1, regardless of the presence of interleukin-6 or bone marrow stromal cells. Conversely, the lysosomal vacuolar H+-ATPase inhibitor blocks SAR-induced LCD. SAR further upregulates reactive oxygen species. Pom enhances SAR-induced direct and indirect killing even in MM cells resistant to Pom/Len. Taken together, SAR is the first therapeutic monoclonal antibody mediating direct cytotoxicity against MM cells via multiple mechanisms of action. Our data show that Pom augments both direct and effector cell-mediated MM cytotoxicity of SAR, providing the framework for combination clinical trials.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Anticorpos Monoclonais Humanizados/farmacologia , Apoptose/efeitos dos fármacos , Lisossomos/fisiologia , Glicoproteínas de Membrana/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Talidomida/análogos & derivados , ADP-Ribosil Ciclase 1/fisiologia , Actinas/química , Genes p53/fisiologia , Humanos , Glicoproteínas de Membrana/fisiologia , Microdomínios da Membrana/fisiologia , Mieloma Múltiplo/patologia , Espécies Reativas de Oxigênio/metabolismo , Talidomida/farmacologiaRESUMO
Carcinogenesis is most often viewed as a multistage disease process. An exception to this was suggested for neu transformation of mammary cells in a transgenic model (Muller et al., 1988); however, this interpretation is controversial (Bouchard et al., 1989). In order to better define neu mammary transformation in vivo, we directly measured the genetic penetrance of the neu oncogene. Mammary cells in situ were infected with replication-defective retroviral vectors carrying the activated neu oncogene (pJRneu). A limiting dilution in vivo transplantation assay was used to measure the percentage of mammary clonogenic (stem-like) cells that stably and functionally integrated this vector. Based on this, the percentage of clonogens integrating and expressing neu that could progress to mammary carcinomas was quantified to estimate the penetrance of this gene in mammary carcinogenesis. The genetic penetrance of neu was 3.6% (95% confidence interval 2.2%-5.8%). This high degree of genetic penetrance is compatible with the observations that certain neu-transgenic mice develop a very great number of mammary carcinomas (Muller et al., 1988). However, whether these data are compatible with a single-step transformation model (100% penetrance) is uncertain and is discussed.
Assuntos
Transformação Celular Neoplásica/genética , Genes erbB-2 , Neoplasias Mamárias Experimentais/genética , Animais , Divisão Celular/genética , Feminino , Vetores Genéticos , Neoplasias Mamárias Experimentais/patologia , Camundongos , RNA Neoplásico/genética , Ratos , Ratos Wistar , Retroviridae/genéticaRESUMO
Although paclitaxel is an important chemotherapy agent for the treatment of patients with epithelial ovarian cancer, its utility is significantly limited by the frequent development of drug resistance. Recent evidence suggests that resistance to chemotherapy may be partly related to defects in the apoptotic pathway. In this study we have investigated whether enhancement of apoptotic pathway function through stable expression of the BAD protein is capable of sensitizing human epithelial ovarian cancer cells to the effects of chemotherapy. Expression of HA-BAD in six separate clonal transfectants from two different ovarian cancer cell lines was found to significantly enhance the cytotoxic effects of paclitaxel, vincristine, and, to a lesser extent, etoposide. Enhancement of paclitaxel-induced apoptosis in HA-BAD-expressing clones was demonstrated by trypan blue exclusion, clonogenic cell assay, and flow cytometric evaluation. Importantly, this effect was associated with binding of HA-BAD to BCL-xL and concomitant disruption of BAX:BCL-xL interaction. Taken together, these data suggest that the development of small molecules which mimic the effects of BAD may represent a new class of drugs capable of preventing or reversing resistance to chemotherapy agents such as paclitaxel.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/fisiologia , Proteínas de Transporte/fisiologia , Resistencia a Medicamentos Antineoplásicos , Oncogenes , Neoplasias Ovarianas/patologia , Paclitaxel/farmacologia , Animais , Líquido Ascítico/citologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Células Clonais/efeitos dos fármacos , DNA de Neoplasias/análise , Células Epiteliais/metabolismo , Feminino , Humanos , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Ovário/citologia , Ovário/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína de Morte Celular Associada a bclRESUMO
PURPOSE: Expression of the pro-apoptotic protein BAX sensitizes ovarian cancer cell lines to paclitaxel in vitro by enhancing the pathway of programmed cell death. The present study was performed to determine the relationship between BAX expression and clinical outcome in 45 patients with newly diagnosed ovarian cancer. METHODS: BAX protein expression was analyzed by immunohistochemistry, and its relationship with clinical outcome was determined. Assessment of BAX mRNA transcript levels and mutational analysis of the BAX coding region were also performed. RESULTS: BAX protein was expressed at high levels (defined as > or = 50% of tumor cells positive) in tumor tissue from 60% of newly diagnosed patients. All patients whose tumors expressed high levels of BAX achieved a complete response (CR) to first-line chemotherapy that contained paclitaxel plus a platinum analogue, compared with 57% of patients in the low-BAX group (P = .036). After a median follow-up of 1.9 years, the median disease-free survival (DFS) of patients in the high-BAX group has not been reached, compared with a median DFS of 1.1 years for low-BAX expressors (P = .0061). BAX retained independent prognostic significance in multivariate analysis when corrected for stage and histology. BAX mRNA transcripts were easily detected in samples with low BAX protein expression, and no BAX mutations were identified. CONCLUSION: The correlation between high BAX levels and improved clinical outcome suggests that an intact apoptotic pathway is an important determinant of chemoresponsiveness in ovarian cancer patients who receive paclitaxel.
Assuntos
Carcinoma/patologia , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas/análise , Antineoplásicos Fitogênicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/química , DNA Complementar/genética , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neoplasias Ovarianas/química , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/administração & dosagem , Prognóstico , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , Análise de Sequência de DNA , Proteína X Associada a bcl-2RESUMO
Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein-Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 microg/ml) and anti-human IL-6 (10 microg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 microM) and its immunomodulatory analog IMiD1-CC4047 (1 microM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal-MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Mieloma Múltiplo/patologia , Células Estromais/citologia , Inibidores da Angiogênese/farmacologia , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Adesão Celular , Comunicação Celular/fisiologia , Técnicas de Cocultura , Interações Medicamentosas , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Interleucina-6/metabolismo , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/fisiopatologia , Células Estromais/metabolismo , Células Estromais/fisiologia , Talidomida/farmacologia , Células Tumorais Cultivadas , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Kaposi's sarcoma-associated herpes virus (KSHV) is associated with Kaposi's sarcoma, multicentric Castleman's disease, and body cavity-based lymphomas, settings in which human interleukin-6 (hIL-6) acts as a growth factor. The KSHV open reading frame K2 encodes for viral IL-6 (vIL-6), a protein with 25% amino acid identity to hIL-6, which can promote the growth of hIL-6-dependent cell lines. In the present study, we characterized biological sequelae and signaling cascades triggered by hIL-6 versus vIL-6 in the hIL-6-dependent MH60 and B9 cell lines. Both hIL-6 and vIL-6 induced significant increases (P < 0.01) in DNA synthesis in these cell lines in a dose-dependent fashion. Neutralizing anti-hIL-6 antibody (Ab) inhibited DNA synthesis triggered by hIL-6, without similarly affecting proliferation in response to vIL-6. On the other hand, antimouse IL-6 receptor (mIL-6R) Ab blocked response to vIL-6, but not that to hIL-6. Both hIL-6 and vIL-6 activated gp130, Janus kinase 1, signal transducers and activators of transcription-3, and mitogen-activated protein kinase in both MH60 and B9 cells. Proliferation of these cell lines in response to both hIL-6 and vIL-6 was blocked by PD98059, an inhibitor of MEK1 activation. These data suggest that MEK1 activation mediates the proliferative response to both cytokines. Finally, both hIL-6 and vIL-6 also maintained viability of serum-starved MH60 and B9 cells and blocked dexamethasone-induced apoptosis of MM.1S human myeloma cells. Further characterization of the signaling cascades mediating the growth and antiapoptotic effects of vIL-6 versus hIL-6 may help identify their unique roles in disease pathogenesis in Kaposi's sarcoma and other KSHV-associated neoplasms.
Assuntos
Interleucina-6/farmacologia , Proteínas Proto-Oncogênicas , Transdução de Sinais/efeitos dos fármacos , Proteínas Virais/farmacologia , Animais , Anticorpos/farmacologia , Antígenos CD/efeitos dos fármacos , Antígenos CD/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Receptor gp130 de Citocina , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Herpesvirus Humano 8/genética , Humanos , Hibridomas , Interleucina-6/genética , Interleucina-6/imunologia , Janus Quinase 1 , Janus Quinase 2 , MAP Quinase Quinase 1 , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Testes de Neutralização , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Receptores de Interleucina-6/imunologia , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Transativadores/efeitos dos fármacos , Transativadores/metabolismo , Células Tumorais Cultivadas , Tirosina/metabolismo , Proteínas Virais/genéticaRESUMO
Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide (Len) and pomalidomide trigger anti-tumor activities in multiple myeloma (MM) by targetting cereblon and thereby impacting IZF1/3, c-Myc and IRF4. Histone deacetylase inhibitors (HDACi) also downregulate c-Myc. We therefore determined whether IMiDs with HDACi trigger significant MM cell growth inhibition by inhibiting or downregulating c-Myc. Combination treatment of Len with non-selective HDACi suberoylanilide hydroxamic acid or class-I HDAC-selective inhibitor MS275 induces synergic cytotoxicity, associated with downregulation of c-Myc. Unexpectedly, we observed that decreased levels of cereblon (CRBN), a primary target protein of IMiDs, was triggered by these agents. Indeed, sequential treatment of MM cells with MS275 followed by Len shows less efficacy than simultaneous treatment with this combination. Importantly ACY1215, an HDAC6 inhibitor with minimal effects on class-I HDACs, together with Len induces synergistic MM cytotoxicity without alteration of CRBN expression. Our results showed that only modest class-I HDAC inhibition is able to induce synergistic MM cytotoxicity in combination with Len. These studies may provide the framework for utilizing HDACi in combination with Len to both avoid CRBN downregulation and enhance anti-MM activities.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Inibidores de Histona Desacetilases/administração & dosagem , Imunomodulação , Mieloma Múltiplo/tratamento farmacológico , Sinergismo Farmacológico , Citometria de Fluxo , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Immunoblotting , Técnicas In Vitro , Lenalidomida , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Transfecção , VorinostatRESUMO
Histone deacetylase (HDAC) inhibitors have been extensively investigated as therapeutic agents in cancer. However, the biological role of class IIa HDACs (HDAC4, 5, 7 and 9) in cancer cells, including multiple myeloma (MM), remains unclear. Recent studies show HDAC4 interacts with activating transcription factor 4 (ATF4) and inhibits activation of endoplasmic reticulum (ER) stress-associated proapoptotic transcription factor C/EBP homologous protein (CHOP). In this study, we hypothesized that HDAC4 knockdown and/or inhibition could enhance apoptosis in MM cells under ER stress condition by upregulating ATF4, followed by CHOP. HDAC4 knockdown showed modest cell growth inhibition; however, it markedly enhanced cytotoxicity induced by either tunicamycin or carfilzomib (CFZ), associated with upregulating ATF4 and CHOP. For pharmacological inhibition of HDAC4, we employed a novel and selective class IIa HDAC inhibitor TMP269, alone and in combination with CFZ. As with HDAC4 knockdown, TMP269 significantly enhanced cytotoxicity induced by CFZ in MM cell lines, upregulating ATF4 and CHOP and inducing apoptosis. Conversely, enhanced cytotoxicity was abrogated by ATF4 knockdown, confirming that ATF4 has a pivotal role mediating cytotoxicity in this setting. These results provide the rationale for novel treatment strategies combining class IIa HDAC inhibitors with ER stressors, including proteasome inhibitors, to improve patient outcome in MM.
Assuntos
Estresse do Retículo Endoplasmático , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Mieloma Múltiplo/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Técnicas de Silenciamento de Genes , Histona Desacetilases/genética , Humanos , Interleucina-6/metabolismo , Isoenzimas , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Inibidores de Proteassoma/farmacologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
We developed a simple and rapid method to enrich tumor cells within bone marrow (BM) aspirates from patients with multiple myeloma (MM). Thirty patients with a median of 50% (8-85%) MM cells by morphology and 55% (6--85%) MM cells identified by CD38+CD45-cell surface phenotype were studied. BM mononuclear cells (BMMCs) were isolated by Ficoll Hypaque sedimentation and incubated with a cocktail of mouse monoclonal antibodies (mAbs) directed against CD3 (T cells); CD11b and CD14 (monocytes); CD33 (myeloid cells), CD45 and CD45RA (leucocyte common antigen); CD32 as well as glycophorin A. After the addition of anti-mouse Fc Ig-coated immunomagnetic beads, mAb-bound cells were removed in a magnetic field. The residual cell populations were enriched for MM cells, evidenced by >95% plasma cell morphology and >95% CD38+CD45RA-cell surface phenotype. Since this method requires only two short incubations, cell losses were minimal and the yield of MM cells was therefore high (>95%). Viability of the MM-cell enriched fractions was 99%, and these cells were functional in assays of proliferation, cell cycle analysis and immunoglobulin secretion. This immunomagnetic bead depletion method therefore permits the ready isolation of homogeneous populations of patient MM cells for use in both cellular and molecular studies.