ABSTRACT
The benefit of adding the antiangiogenic drug aflibercept to FOLFIRI regime in metastatic colorectal cancer (CRC) patients resistant to or progressive on an oxaliplatin-based therapy has been previously demonstrated. However, the absence of validated biomarkers to predict greater outcomes is a major challenge encountered when using antiangiogenic therapies. In this study we investigated profiles of circulating microRNAs (miRNAs) to build predictive models of response to treatment and survival. Plasma was obtained from 98 metastatic CRC patients enrolled in a clinical phase II trial before receiving FOLFIRI plus aflibercept treatment, and the circulating levels of 754 individual miRNAs were quantified using real-time PCR. A distinct signature of circulating miRNAs differentiated responder from non-responder patients. Remarkably, most of these miRNAs were found to target genes that are involved in angiogenic processes. Accordingly, some of these miRNAs had predictive value and entered in predictive models of response to therapy, progression of disease, and survival of patients treated with FOLFIRI plus aflibercept. Among these miRNAs, circulating levels of hsa-miR-33b-5p efficiently discriminated between responder and non-responder patients and predicted the risk of disease progression. Moreover, the combination of circulating VEGF-A and miR-33b-5p levels improved clinical stratification of metastatic CRC patients who were to receive FOLFIRI plus aflibercept treatment. In conclusion, our study supports circulating miRNAs as valuable biomarkers for predicting better outcomes in metastatic CRC patients treated with FOLFIRI plus aflibercept.
Subject(s)
Circulating MicroRNA , Colonic Neoplasms , Colorectal Neoplasms , MicroRNAs , Rectal Neoplasms , Humans , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Camptothecin , Fluorouracil , Leucovorin/therapeutic use , Leucovorin/adverse effects , Receptors, Vascular Endothelial Growth Factor , Recombinant Fusion Proteins/therapeutic use , Colonic Neoplasms/drug therapy , Rectal Neoplasms/drug therapy , MicroRNAs/genetics , MicroRNAs/therapeutic use , Antineoplastic Combined Chemotherapy ProtocolsSubject(s)
Electrophoresis, Polyacrylamide Gel/methods , Lyases/analysis , N-Glycosyl Hydrolases/analysis , 8-Hydroxy-2'-Deoxyguanosine , Arabidopsis/enzymology , DNA Glycosylases , DNA Repair/genetics , DNA Repair/physiology , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Escherichia coli/enzymology , Luminescent Measurements , Substrate SpecificityABSTRACT
Repair of the ubiquitous mutagenic lesion 7,8-dihydro-8-oxoguanine (8-oxoG) is initiated in eukaryotes by DNA glycosylases/lyases, such as yeast Ogg1, that do not share significant sequence identity with their prokaryotic counterparts, typified by Escherichia coli MutM (Fpg) protein. The unexpected presence of a functional mutM orthologue in the model plant Arabidopsis thaliana has brought into question the existence of functional OGG1 orthologues in plants. We report here the cDNA cloning, expression and functional characterization of AtOGG1, an Arabidopsis thaliana gene widely expressed in different plant tissues which encodes a 40.3 kDa protein with significant sequence identity to yeast and human Ogg1 proteins. Purified AtOgg1 enzyme specifically cleaves duplex DNA containing an 8-OxoG:C mispair, and the repair reaction proceeds through an imine intermediate
Subject(s)
Arabidopsis/genetics , Escherichia coli Proteins , Guanine/analogs & derivatives , N-Glycosyl Hydrolases/genetics , Amino Acid Sequence , Arabidopsis/enzymology , Bacterial Proteins/genetics , DNA Glycosylases , DNA, Complementary/chemistry , DNA, Complementary/genetics , DNA-Formamidopyrimidine Glycosylase , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Genetic Complementation Test , Guanine/metabolism , Molecular Sequence Data , Mutation , N-Glycosyl Hydrolases/metabolism , Phenotype , RNA, Plant/genetics , RNA, Plant/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Substrate Specificity , Tissue DistributionABSTRACT
Reactive oxygen species (ROS) are ubiquitous DNA-damaging agents, and the repair of oxidative DNA lesions is essential to prevent mutations and cell death. Escherichia coli endonuclease III is the prototype repair enzyme for removal of oxidized pyrimidines from DNA. A database homology search identified a genomic sequence in Arabidopsis thaliana encoding a predicted protein with sequence similarity to E. coli endonuclease III. We cloned, sequenced and expressed the corresponding cDNA, which encodes a 39.1 kDa protein containing several sequence motifs conserved in endonuclease III homologues, including an iron-sulfur cluster domain and critical residues at the active site. The protein, designated AtNTH1, was over-expressed in E. coli and purified to apparent homogeneity. AtNTH1 exhibits DNA-glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polydeoxyribonucleotides. The enzyme also possesses an apurinic/apyrimidinic lyase activity on UV- and gamma-irradiated DNA substrates. The AtNTH1 gene contains 10 introns and 11 exons and is widely expressed in different plant tissues. Our results suggest that AtNTH1 is a structural and functional homologue of endonuclease III and probably plays a major role in plant defence against oxidative DNA damage.
