RESUMEN
To identify new candidate cancer drug targets, we used RNAi as a tool to functionally evaluate genes that play a role in maintaining human tumor cell survival. We screened a small interfering RNA (siRNA) library directed against approximately 3,700 individual genes to assess the ability of siRNAs to induce cell death in an in vitro cell cytotoxicity assay. We found that siRNAs specifically targeting ras-related nuclear protein (Ran), targeting protein for Xenopus kinesin-like protein 2 (TPX2), and stearoyl-CoA desaturase 1 (SCD1), significantly reduced the survival of multiple human tumor cell lines. Further target validation studies revealed that treatment with Ran and TPX2 siRNAs differentially reduced the survival of activated K-Ras-transformed cells compared with their normal isogenic counterparts in which the mutant K-Ras gene had been disrupted (DKS-8). Knockdown of Ran and TPX2 in activated mutant K-Ras cells selectively induced S-phase arrest or transient G(2)-M arrest phenotypes, respectively, that preceded apoptotic cell death. Given our observations that Ran and TPX2 depletion preferentially reduces the survival of activated K-Ras-transformed cells, these two proteins may serve as useful anticancer targets in tumors expressing the activated K-Ras oncogene.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , ARN Interferente Pequeño/genética , Estearoil-CoA Desaturasa/genética , Proteínas de Xenopus/genética , Proteína de Unión al GTP ran/genética , Ciclo Celular/genética , Muerte Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Biblioteca de Genes , Genes ras , Humanos , Neoplasias/patología , Interferencia de ARNRESUMEN
ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.
Asunto(s)
Compuestos de Bifenilo/farmacología , Carcinoma de Células Pequeñas/tratamiento farmacológico , Carcinoma de Células Pequeñas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Nitrofenoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Compuestos de Bifenilo/administración & dosificación , Carboplatino/administración & dosificación , Carcinoma de Células Pequeñas/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Regulación hacia Abajo , Sinergismo Farmacológico , Etopósido/administración & dosificación , Humanos , Neoplasias Pulmonares/patología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Nitrofenoles/administración & dosificación , Piperazinas/administración & dosificación , Piperazinas/farmacología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Interferente Pequeño/genética , Sulfonamidas/administración & dosificación , Transfección , Regulación hacia ArribaRESUMEN
To identify cancer-specific targets, we have conducted a synthetic lethal screen using a small interfering RNA (siRNA) library targeting approximately 4,000 individual genes for enhanced killing in the DLD-1 colon carcinoma cell line that expresses an activated copy of the K-Ras oncogene. We found that siRNAs targeting baculoviral inhibitor of apoptosis repeat-containing 5 (survivin) significantly reduced the survival of activated K-Ras-transformed cells compared with its normal isogenic counterpart in which the mutant K-Ras gene had been disrupted (DKS-8). In addition, survivin siRNA induced a transient G(2)-M arrest and marked polyploidy that was associated with increased caspase-3 activation in the activated K-Ras cells. These results indicate that tumors expressing the activated K-Ras oncogene may be particularly sensitive to inhibitors of the survivin protein.
Asunto(s)
Transformación Celular Neoplásica/patología , Genes ras , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/metabolismo , Alelos , Apoptosis , Muerte Celular , Supervivencia Celular , Células Clonales , Fase G2 , Genes Relacionados con las Neoplasias , Humanos , Proteínas Inhibidoras de la Apoptosis , Mitosis , Proteínas Mutantes/metabolismo , Poliploidía , ARN Interferente Pequeño/metabolismo , SurvivinRESUMEN
Successful siRNA therapeutics requires the optimal integration of multiple components, including an efficient delivery system, a disease indication that is appropriate for siRNA-based therapy, and a potent and nontoxic siRNA against a robust therapeutic target. Although all currently available delivery systems have limitations, it is important to recognize that a careful selection of the disease indication, therapeutic target, and siRNA molecule could partially compensate for deficiencies associated with the delivery system and makes it possible to advance a therapeutic siRNA regimen. In this study, we present the development of siRNA therapeutics for hepatocellular carcinoma using an integrated approach, including the development of an efficient lipid nanoparticle delivery system, the identification of a robust therapeutic target that does not trigger liver toxicity upon target knockdown, and the selection of potent and nonimmunogenic siRNA molecules against the target. The resulting siRNA-containing lipid nanoparticles produced significant antitumor efficacy in orthotopic hepatocellular carcinoma models, and, thus, represent a promising starting point for the development of siRNA therapeutics for hepatocellular carcinoma.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Neoplasias Hepáticas/tratamiento farmacológico , Nanopartículas , ARN Interferente Pequeño/administración & dosificación , Animales , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Femenino , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas Experimentales , Ratones , Ratones SCID , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/toxicidadRESUMEN
Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.
Asunto(s)
Expresión Génica , ARN Interferente Pequeño/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The BGL2 gene encodes a unique 1,3-beta-glucosyltransferase (Bgl2p) present in the cell wall of Candida albicans and other fungi. Although believed to be involved in cell wall assembly, disruption of the gene in saccharomyces cerevisiae showed no apparent phenotype. We performed sequential disruptions of the BGL2 loci in a homozygous ura3 clinical isolate of C. albicans using the URA3 blaster method, in order to investigate the role of Bgl2p in this dimorphic, pathogenic fungus. Strain CACW-1 contained disruptions of both homologues of the BGL2 gene and lacked Bgl2p, as assessed by protein extraction, SDS-PAGE and Western blot analysis, and enzyme assay; however, residual non-Bgl2p transferase activity was detected. CACW-1 was attenuated in virulence for mice when compared to an isogenic parent strain, and fewer organisms were recovered from the kidneys of infected animals. Additional phenotypic changes included: (1) a dramatic increase in the sensitivity to the chitin synthesis inhibitor nikkomycin Z when CACW-1 cells were incubated at 37 or 42 degrees C; (2) an 8.7 +/- 1.6% slower growth rate at 37 degrees C for CACW-1 when compared to its isogenic parent; and (3) aggregation of CACW-1 cells during stationary phase and/or incubation of stationary phase cells in phosphate buffer. Characterization of SDS-extracted cell walls did not reveal any significant differences in the levels of 1,3-beta- or 1,6-beta-glucan. These data reveal that loss of Bgl2p does have a phenotype in C. albicans, and indicate that (1) loss of Bgl2p function renders cells more dependent on chitin for wall integrity, and attenuates virulence (probably due to subtle changes in wall structure), and (2) that additional 1,3-beta-glucosyltransferases are present in the C. albicans BGL2 disruptant.
Asunto(s)
Candida albicans/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Glucano Endo-1,3-beta-D-Glucosidasa/genética , Glucosiltransferasas/genética , Animales , Antifúngicos/farmacología , Candida albicans/enzimología , Candida albicans/patogenicidad , Candidiasis , Pared Celular/química , Pared Celular/metabolismo , Pared Celular/ultraestructura , Glucanos/química , Glicosilación , Riñón/microbiología , Ratones , Pruebas de Sensibilidad Microbiana , Mutagénesis , Fenotipo , Virulencia/genéticaRESUMEN
Survivin, one of the most tumor-specific gene products, has been implicated in both anti-apoptosis and cytokinesis. However, the mechanism by which survivin regulates these two different processes is still elusive. Here, we show that survivin binds to the catalytic domain of Aurora-B. We demonstrate that in the presence of survivin, Aurora-B phosphorylates histone H3 much more efficiently than in the absence of survivin in a cell-free system. Furthermore, we confirm that cells lacking survivin due to survivin antisense oligonucleotide-treatment have lower Aurora-B kinase activity, whereas cells overexpressing survivin have higher Aurora-B kinase activity. We also provide evidence that depletion of survivin by survivin antisense oligonucleotide treatment causes significant reduction of endogenous phosphorylated histone H3 and mislocalization of Aurora-B. These results indicate that survivin stimulates Aurora-B kinase activity and helps correctly target Aurora-B to its substrates during the cell cycle, thus providing a mechanism as to how survivin exerts its function in human cells.