RESUMEN
Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease FEN1, was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through in vitro biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in CDC4, a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between FEN1 and MRE11A, another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model.
Asunto(s)
Inestabilidad Cromosómica , Neoplasias Colorrectales/genética , Endonucleasas de ADN Solapado , Redes Reguladoras de Genes , Evolución Biológica , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas de ADN Solapado/antagonistas & inhibidores , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Genes Letales , Genes Sintéticos , Humanos , Proteína Homóloga de MRE11 , Terapia Molecular Dirigida , Mutación , ARN Interferente Pequeño/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
OBJECTIVE: Mosaicism with trisomy confined to the placenta is present in ~1% of ongoing pregnancies at the time of chorionic villus sampling. Some studies have found reduced fetal growth in confined placental trisomy. The objective of this study was to assess placental weight and feto-placental weight ratio in pregnancies with trisomy confined to the placenta, and to correlate them with the level of trisomy in the three major placental lineages. METHODS: We conducted a retrospective study of 69 pregnancies with prenatally diagnosed mosaic trisomy in which the trisomic cells were confined to the placenta. Placental weight and feto-placental weight ratio were compared to those of matched controls, and placental weight was also analyzed for associations with the type and level of trisomy. Placental pathology was also reviewed. RESULTS: The pregnancies with mosaic trisomy were found to have lower placental weights than matched controls, but normal feto-placental weight ratios. Placental weight was not associated with the type or level of trisomic cells in the three placental lineages at term (chorionic plate, chorionic villus mesenchyme, and trophoblast). There were no pathognomonic findings on routine placental pathology of the trisomic placentas. CONCLUSION: Although placental weight was reduced (with normal feto-placental weight ratio) in pregnancies with trisomy confined to the placenta, the level of placental trisomy was not correlated with placental weight. Thus, trisomy may alter placental function rather than have a direct hypoplastic effect on placental growth. More in-depth studies beyond routine pathology are required to identify how trisomy affects placental function.
Asunto(s)
Placenta/patología , Trisomía/patología , Estudios de Casos y Controles , Femenino , Peso Fetal , Humanos , Tamaño de los Órganos , Embarazo , Estudios RetrospectivosRESUMEN
Mutations that cause chromosome instability (CIN) in cancer cells produce "sublethal" deficiencies in an essential process (chromosome segregation) and, therefore, may represent a major untapped resource that could be exploited for therapeutic benefit in the treatment of cancer. If second-site unlinked genes can be identified, that when knocked down, cause a synthetic lethal (SL) phenotype in combination with a somatic mutation in a CIN gene, novel candidate therapeutic targets will be identified. To test this idea, we took a cross species SL candidate gene approach by recapitulating a SL interaction observed between rad54 and rad27 mutations in yeast, via knockdown of the highly sequence- and functionally-related proteins RAD54B and FEN1 in a cancer cell line. We show that knockdown of RAD54B, a gene known to be somatically mutated in cancer, causes CIN in mammalian cells. Using high-content microscopy techniques, we demonstrate that RAD54B-deficient human colorectal cancer cells are sensitive to SL killing by reduced FEN1 expression, while isogenic RAD54B proficient cells are not. This conserved SL interaction suggests that extrapolating SL interactions observed in model organisms for homologous genes mutated in human cancers will aid in the identification of novel therapeutic targets for specific killing of cancerous cells exhibiting CIN.
Asunto(s)
Apoptosis , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , ADN Helicasas/deficiencia , ADN Helicasas/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Silenciador del Gen , Proteínas Nucleares/deficiencia , Proteínas Nucleares/metabolismo , Inestabilidad Cromosómica/genética , Neoplasias Colorrectales/genética , ADN Helicasas/genética , Endonucleasas de ADN Solapado/genética , Humanos , Proteínas Nucleares/genética , Fenotipo , Especificidad por Sustrato , Técnicas de Cultivo de TejidosRESUMEN
OBJECTIVE: To investigate presence of trisomy in amniotic epithelium (uncultured amnion) and mesenchyme (cultured amnion) from mosaic cases to understand the origins of these tissues and their relationship to pregnancy outcome. METHODS: Polymerase chain reaction (PCR) of microsatellite loci was used to determine the presence of trisomy (of meiotic origin only) in amnion samples from 33 placentas previously ascertained because of a prenatal diagnosis of trisomy mosaicism that was predominantly confined to the placental tissues. RESULTS: In 16 (48%) of 33 cases, trisomy was confirmed to be present by molecular analysis of uncultured amnion. In contrast, cytogenetic analysis of cultured amnion showed trisomy in only 2 of 20 informative cases. The molecular detection of trisomy in amnion was strongly associated with poor pregnancy outcome (intrauterine growth restriction, fetal anomalies and/or intrauterine/neonatal death) even when analysis was limited to cases negative for the trisomy on amniotic fluid (N = 22, p = 0.0005). CONCLUSIONS: We infer that amniotic mesenchyme (usually diploid) derives from early embryonic mesoderm of the primitive streak and not from the hypoblast as is commonly cited. Trisomy in amniotic epithelium suggests that high numbers of abnormal cells were present in the epiblast, and this correlates with poor outcome even when the subsequently derived fetus and amniotic mesenchyme appear to carry only diploid cells.