RESUMO
The identification of recurrent somatic mutations in genes encoding epigenetic enzymes has provided a strong rationale for the development of compounds that target the epigenome for the treatment of cancer. This notion is supported by biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases to promoter regions through association with oncogenic fusion proteins such as PML-RARα and AML1-ETO. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis; however, it remains unclear why tumor cells are more sensitive to HDACi-induced cell death than normal cells. Herein, we assessed the biological and molecular responses of isogenic normal and transformed cells to the FDA-approved HDACi vorinostat and romidepsin. Both HDACi selectively killed cells of diverse tissue origin that had been transformed through the serial introduction of different oncogenes. Time-course microarray expression profiling revealed that normal and transformed cells transcriptionally responded to vorinostat treatment. Over 4200 genes responded differently to vorinostat in normal and transformed cells and gene ontology and pathway analyses identified a tumor-cell-selective pro-apoptotic gene-expression signature that consisted of BCL2 family genes. In particular, HDACi induced tumor-cell-selective upregulation of the pro-apoptotic gene BMF and downregulation of the pro-survival gene BCL2A1 encoding BFL-1. Maintenance of BFL-1 levels in transformed cells through forced expression conferred vorinostat resistance, indicating that specific and selective engagement of the intrinsic apoptotic pathway underlies the tumor-cell-selective apoptotic activities of these agents. The ability of HDACi to affect the growth and survival of tumor cells whilst leaving normal cells relatively unharmed is fundamental to their successful clinical application. This study provides new insight into the transcriptional effects of HDACi in human donor-matched normal and transformed cells, and implicates specific molecules and pathways in the tumor-selective cytotoxic activity of these compounds.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores de Histona Desacetilases/toxicidade , Histona Desacetilases/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Depsipeptídeos/toxicidade , Epigenômica , Regulação da Expressão Gênica , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/toxicidade , Antígenos de Histocompatibilidade Menor , Neoplasias/metabolismo , Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , VorinostatRESUMO
There is significant interest in treating cancers by blocking protein synthesis, to which hematological malignancies seem particularly sensitive. The translation elongation inhibitor homoharringtonine (Omacetaxine mepesuccinate) is undergoing clinical trials for chronic myeloid leukemia, whereas the translation initiation inhibitor silvestrol has shown promise in mouse models of cancer. Precisely how these compounds induce cell death is unclear, but reduction in Mcl-1, a labile pro-survival Bcl-2 family member, has been proposed to constitute the critical event. Moreover, the contribution of translation inhibitors to neutropenia and lymphopenia has not been precisely defined. Herein, we demonstrate that primary B cells and neutrophils are highly sensitive to translation inhibitors, which trigger the Bax/Bak-mediated apoptotic pathway. However, contrary to expectations, reduction of Mcl-1 did not significantly enhance cytotoxicity of these compounds, suggesting that it does not have a principal role and cautions that strong correlations do not always signify causality. On the other hand, the killing of T lymphocytes was less dependent on Bax and Bak, indicating that translation inhibitors can also induce cell death via alternative mechanisms. Indeed, loss of clonogenic survival proved to be independent of the Bax/Bak-mediated apoptosis altogether. Our findings warn of potential toxicity as these translation inhibitors are cytotoxic to many differentiated non-cycling cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Harringtoninas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Triterpenos/farmacologia , Animais , Células Cultivadas , Células HL-60 , Mepesuccinato de Omacetaxina , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neutrófilos/efeitos dos fármacos , Elongação Traducional da Cadeia Peptídica/efeitos dos fármacos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/antagonistas & inibidores , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis, and modulation of immune responses, and show promising activity against hematological malignancies in clinical trials. Using the Emu-myc model of B cell lymphoma, we screened tumors with defined genetic alterations in apoptotic pathways for therapeutic responsiveness to the HDACi vorinostat. We demonstrated a direct correlation between induction of tumor cell apoptosis in vivo and therapeutic efficacy. Vorinostat did not require p53 activity or a functional death receptor pathway to kill Emu-myc lymphomas and mediate a therapeutic response but depended on activation of the intrinsic apoptotic pathway with the proapoptotic BH3-only proteins Bid and Bim playing an important role. Our studies provide important information regarding the mechanisms of action of HDACi that have broad implications regarding stratification of patients receiving HDACi therapy alone or in combination with other anticancer agents.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Proteína 11 Semelhante a Bcl-2 , Genes myc , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Vorinostat , Proteína bcl-X/fisiologiaRESUMO
Mice lacking suppressor of cytokine signaling 3 (SOCS3) exhibited embryonic lethality with death occurring between days 11 and 13 of gestation. At this stage, SOCS3(-/-) embryos were slightly smaller than wild type but appeared otherwise normal, and histological analysis failed to detect any anatomical abnormalities responsible for the lethal phenotype. Rather, in all SOCS3(-/-) embryos examined, defects were evident in placental development that would account for their developmental arrest and death. The placental spongiotrophoblast layer was significantly reduced and accompanied by increased numbers of giant trophoblast cells. Delayed branching of the chorioallantois was evident, and, although embryonic blood vessels were present in the labyrinthine layer of SOCS3(-/-) placentas, the network of embryonic vessels and maternal sinuses was poorly developed. Yolk sac erythropoiesis was normal, and, although the SOCS3(-/-) fetal liver was small at day 12.5 of gestation (E12.5), normal frequencies of erythroblasts and hematopoietic progenitor cells, including blast forming unit-erythroid (BFU-E) and, colony forming unit-erythroid (CFU-E) were present at both E11.5 and E12.5. Colony formation for both BFU-E and CFU-E from SOCS3(-/-) mice displayed wild-type quantitative responsiveness to erythropoietin (EPO), in the presence or absence of IL-3 or stem cell factor (SCF). These data suggest that SOCS3 is required for placental development but dispensable for normal hematopoiesis in the mouse embryo.