RESUMEN
The genetic concept of synthetic lethality provides a framework for identifying genotype-selective anticancer agents. In this approach, changes in cellular physiology that arise as a consequence of oncogene activation or tumor suppressor gene loss, rather than oncoproteins themselves, are targeted to achieve tumor selectivity. Here we show that agonists of the TRAIL death receptor DR5 potently induce apoptosis in human cells overexpressing the MYC oncogene, both in vitro and as tumor xenografts in vivo. MYC sensitizes cells to DR5 in a p53-independent manner by upregulating DR5 cell surface levels and stimulating autocatalytic processing of procaspase-8. These results identify a novel mechanism by which MYC sensitizes cells to apoptosis and validate DR5 agonists as potential MYC-selective cancer therapeutics.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis , Mutación/genética , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Interferente Pequeño/farmacología , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Animales , Caspasa 8 , Caspasas/metabolismo , Fibroblastos/metabolismo , Marcación de Gen , Humanos , Masculino , Ratones , Ratones Desnudos , Ratones SCID , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Retroviridae/genética , Trasplante Heterólogo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.
Asunto(s)
Antineoplásicos Fitogénicos/metabolismo , Inmunoconjugados/metabolismo , Maitansina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Antineoplásicos Fitogénicos/administración & dosificación , Línea Celular Tumoral , Citoplasma/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Inmunoconjugados/administración & dosificación , Lisosomas/metabolismo , Maitansina/administración & dosificaciónRESUMEN
Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term "clonal dominance". We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780(cis)) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.
Asunto(s)
Transformación Celular Neoplásica , Lentivirus/genética , Animales , Proliferación Celular , Células Clonales/patología , Femenino , Células HCT116 , Humanos , Ratones , ARN Interferente Pequeño/genética , Transducción GenéticaRESUMEN
Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.
Asunto(s)
Neoplasias/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Concentración 50 Inhibidora , Ratones , Neoplasias/genética , Neoplasias/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Interferencia de ARN , Carga Tumoral/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Factor A de Crecimiento Endotelial Vascular/farmacología , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Apoptosis/efectos de los fármacos , Genes myc/fisiología , Genes ras/fisiología , Glicoproteínas de Membrana/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Reguladoras de la Apoptosis , Butadienos/farmacología , Línea Celular Tumoral , Humanos , Nitrilos/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.
Asunto(s)
Neoplasias/enzimología , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras) , Interferencia de ARNRESUMEN
Screening small interfering RNA (siRNA) libraries holds the potential to elucidate gene function as well as discover new targets for the therapeutic treatment of disease. Since the inception of siRNA as a discovery tool, there have been progressive improvements in siRNA design algorithms, the transfection reagents used to deliver them, and the assay formats used to monitor phenotypic changes. These changes have helped to improve the quality of the data emerging from siRNA screens. One variable that introduces inconsistency into high-throughput screening (HTS) of siRNA libraries is the state of the cells used in the assays. Multiple factors can contribute to differences in transfection efficiency as well as the basic cell biology, which can lead to differences in the genes identified in siRNA screens. The authors have developed a system using frozen cell aliquots to use in siRNA HTS, so that a major source of variability introduced into cell-based screens can be standardized. In addition, by transiently transfecting plasmids into cell lines and then freezing these cells down to use in siRNA transfection experiments, they have used this same technology to create new cell lines. This process of using aliquots of frozen cells is logistically advantageous in an HTS setting, as it reduces the time spent maintaining cell lines, as well as reducing possible downtime in screening due to lack of cells or poor cell health.
Asunto(s)
Criopreservación , Biblioteca Genómica , Ensayos Analíticos de Alto Rendimiento/métodos , ARN Interferente Pequeño/metabolismo , Animales , Línea Celular , Supervivencia Celular , Humanos , ARN Interferente Pequeño/genéticaRESUMEN
RNA interference (RNAi) mediated loss-of-function screens have the potential to delineate biological functions of genes and the proteins they encode. RNAi has proven to be a promising technology for identification and validation of new targets for the pharmacological treatment of many diseases including cancer. Here we review the use of high-throughput RNAi screens, examine the types of targets pursued for oncology indications, and discuss the integration of diverse datasets in both target discovery and drug discovery programs.
Asunto(s)
Sistemas de Liberación de Medicamentos , Neoplasias/genética , Interferencia de ARN , Antineoplásicos/farmacología , Diseño de Fármacos , Silenciador del Gen , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/fisiopatologíaRESUMEN
The CDKN2A locus encodes two important tumor suppressors, INK4a and ARF, which respond to oncogenic stresses by inducing cellular senescence. We conducted a genome-scale cDNA overexpression screen using a reporter containing INK4a regulatory sequences to identify novel transcriptional activators of this locus. This screen revealed 285 cDNAs that putatively regulate the transcriptional activation of INK4a. Of these, 56 are annotated as transcription factors, including two previously reported activators of the locus, ETS2 and JUNB. Fourteen genes were further validated for activity and specificity, including several homeodomain proteins. We found that the transcription of one of these, the homeodomain protein MEOX2 (GAX) is enhanced in primary cells during the induction of senescence, and forced expression of this protein results in the induction of premature senescence. We further demonstrate that MEOX2-induced senescence is dependent upon INK4a activity, and chromatin immunoprecipitation studies indicate that MEOX2 directly binds the INK4a promoter. These results support a role for this homeodomain protein as a direct regulator of INK4a transcription and senescence in human cells.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Proteínas de Homeodominio/fisiología , Transactivadores/fisiología , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , ADN Complementario , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The MYC protooncogene is frequently deregulated in human cancers. Here, by screening a kinase-directed library of small inhibitory RNAs, we identify glycogen synthase kinase 3beta (GSK3beta) as a gene whose inactivation potentiates TNF-related apoptosis-inducing ligand death receptor-mediated apoptosis specifically in MYC-overexpressing cells. Small inhibitory RNA-induced silencing of GSK3beta prevents phosphorylation of MYC on T58, thereby inhibiting recognition of MYC by the E3 ubiquitin ligase component FBW7. Attenuating the GSK3beta-FBW7 axis results in stabilization of MYC, up-regulation of surface levels of the TNF-related apoptosis-inducing ligand death receptor 5, and potentiation of death receptor 5-induced apoptosis in vitro and in vivo. These results identify GSK3beta and FBW7 as potential cancer therapeutic targets and MYC as a critical substrate in the GSK3beta survival-signaling pathway. The results also demonstrate paradoxically that MYC-expressing tumors might be treatable by drug combinations that increase rather than decrease MYC oncoprotein function.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Caspasa 8 , Caspasas/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Biblioteca de Genes , Silenciador del Gen , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Glicoproteínas de Membrana/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The p53 tumor-suppressor protein is a critical mediator of cellular growth arrest and the induction of apoptosis. To identify proteins involved in the modulation of p53 transcriptional activity, a gain-of-function cellular screen was carried out with an arrayed matrix of approximately 20,000 cDNAs. Nine genes previously unknown to be involved in regulating p53 activity were identified. Overexpression of seven of these genes (Hey1, Hes1, TFAP4, Osr1, NR2F2, SFRS10, and FLJ11339) resulted in up-regulation of p53 activity; overexpression of two genes (M17S2 and cathepsin B) resulted in down-regulation of p53 activity in mammalian cells. HES1, HEY1, and TFAP4, which are members of the basic helix-loop-helix transcription family, and OSR1 were shown to activate p53 through repression of HDM2 transcription. Ectopic expression of these basic helix-loop-helix transcription factors in both zebrafish and avian developmental systems activated p53 and induced apoptosis in vivo, resulting in a phenotype similar to that of p53 overexpression. Furthermore, ras- and myc-mediated transformation of mouse embryonic fibroblasts was abrogated by expression of HEY1 in a p53-dependent manner. These results suggest that these transcription factors are members of an evolutionarily conserved network that governs p53 function.