RESUMEN
Colorectal cancer is among the leading causes of cancer death in the USA. The polycomb repressive complex 2 (PRC2), including core components SUZ12 and EZH2, represents a key epigenetic regulator of digestive epithelial cell physiology and was previously shown to promote deleterious effects in a number of human cancers, including colon. Using colon cancer stem cells (CCSC) isolated from human primary colorectal tumors, we demonstrate that SUZ12 knockdown and treatment with DZNep, one of the most potent EZH2 inhibitors, increase apoptosis levels, marked by decreased Akt phosphorylation, in CCSCs, while embryonic stem (ES) cell survival is not affected. Moreover, DZNep treatments lead to increased PTEN expression in these highly tumorigenic cells. Taken together, our findings suggest that pharmacological inhibition of PRC2 histone methyltransferase activity may constitute a new, epigenetic therapeutic strategy to target highly tumorigenic and metastatic colon cancer stem cells.
Asunto(s)
Apoptosis , Fosfohidrolasa PTEN/metabolismo , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Animales , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Epigénesis Genética , Técnica del Anticuerpo Fluorescente Indirecta , Células HT29 , Histonas/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias , Fosfohidrolasa PTEN/genética , Fosforilación , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Colorectal cancer is ranked among the top leading causes of cancer death in industrialized populations. Polycomb group proteins, including Suz12 and Ezh2, are epigenetic regulatory proteins that act as transcriptional repressors of many differentiation-associated genes and are overexpressed in a large subset of colorectal cancers. Retinoic acid (RA) acts as a negative regulator of PcG actions in stem cells, but has shown limited therapeutic potential in some solid tumors, including colorectal cancer, in part because of retinoic acid receptor ß silencing. Through treatment with RA, Suz12 shRNA knockdown, or Ezh2 pharmacological inhibition with 3-deazaneplanocin A (DZNep), we increased TRAIL-mediated apoptosis in human colorectal cancer cell lines. This increased apoptosis in human colon cancer cells after RA or DZNep treatment was associated with a ~2.5-fold increase in TNFRSF10B (DR5) transcript levels and a 42% reduction in the H3K27me3 epigenetic mark at the TNFRSF10B promoter after DZNep addition. Taken together, our findings indicate that pharmacological inhibition of Polycomb repressive complex 2 histone methyltransferase activity may constitute a new epigenetic therapeutic strategy to overcome RA non-responsiveness in a subset of colorectal tumors by increasing TRAIL-mediated apoptosis sensitivity.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias del Colon/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Apoptosis/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Epigenómica , Células HT29 , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Células MCF-7 , Proteínas de Neoplasias , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Interferente Pequeño/antagonistas & inhibidores , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Factores de Transcripción , Tretinoina/farmacologíaRESUMEN
Lynch syndrome, also known as hereditary nonpolyposis colon cancer (HNPCC), is the most common known genetic syndrome for colorectal cancer (CRC). MLH1/MSH2 mutations underlie approximately 90% of Lynch syndrome families. A total of 24% of these mutations are missense. Interpreting missense variation is extremely challenging. We have therefore developed multivariate analysis of protein polymorphisms-mismatch repair (MAPP-MMR), a bioinformatic algorithm that effectively classifies MLH1/MSH2 deleterious and neutral missense variants. We compiled a large database (n>300) of MLH1/MSH2 missense variants with associated clinical and molecular characteristics. We divided this database into nonoverlapping training and validation sets and tested MAPP-MMR. MAPP-MMR significantly outperformed other missense variant classification algorithms (sensitivity, 94%; specificity, 96%; positive predictive value [PPV] 98%; negative predictive value [NPV], 89%), such as SIFT and PolyPhen. MAPP-MMR is an effective bioinformatic tool for missense variant interpretation that accurately distinguishes MLH1/MSH2 deleterious variants from neutral variants.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Adenocarcinoma/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Bases de Datos Genéticas , Proteína 2 Homóloga a MutS/genética , Mutación Missense , Proteínas Nucleares/genética , Reparación de la Incompatibilidad de ADN , Femenino , Humanos , Masculino , Inestabilidad de Microsatélites , Análisis Multivariante , Homólogo 1 de la Proteína MutLRESUMEN
The multiresistance transposon Tn1331, which mediates resistance to several aminoglycosides and beta-lactams, includes the aac(6')-Ib, aadA1, bla(OXA-9), and bla(TEM-1) genes. The nucleotide sequence of aac(6')-Ib includes a region identical to that of the bla(TEM-1) gene. This region encompasses the promoter and the initiation codon followed by 15 nucleotides. Since there were three possible translation initiation sites, the amino acid sequence at the N terminus of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] was determined and was found to be SIQHF. This result indicated that aac(6')-Ib includes a translational fusion: the first five amino acids of the leader peptide of the TEM beta-lactamase are fused to the rest of the AAC(6')-Ib protein. This gene fusion could have formed during the genesis of Tn1331 as a consequence of the generation of a 520-nucleotide duplication (M. E. Tolmasky, Plasmid 24:218-226, 1990). An identical gene isolated from a Serratia marcescens strain has been previously described (G. Tran van Nhieu and E. Collatz, J. Bacteriol. 169:5708-5714, 1987). Extraction of the periplasmic proteins of E. coli harboring aac(6')-Ib by spheroplast formation showed that most of the AAC(6')-Ib protein is present in the cytoplasm. A genetic fusion to phoA confirmed these results. AAC(6')-Ib was shown to be evenly distributed inside the cell's cytoplasm by fluorescent microscopy with an AAC(6')-Ib-cyan fluorescent protein fusion.