ABSTRACT
This corrects the article DOI: 10.1038/cdd.2010.65.
ABSTRACT
Glioblastoma is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, survival of patients remains poor. To define novel pathways that regulate susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in glioma, we have performed genome-wide expression profiling of microRNAs (miRs). We show that in TRAIL-resistant glioma cells, levels of different miRs are increased, and in particular, miR-30b/c and -21. We demonstrate that these miRs impair TRAIL-dependent apoptosis by inhibiting the expression of key functional proteins. T98G-sensitive cells treated with miR-21 or -30b/c become resistant to TRAIL. Furthermore, we demonstrate that miR-30b/c and miR-21 target respectively the 3' untranslated region of caspase-3 and TAp63 mRNAs, and that those proteins mediate some of the effects of miR-30 and -21 on TRAIL resistance, even in human glioblastoma primary cells and in lung cancer cells. In conclusion, we show that high expression levels of miR-21 and -30b/c are needed to maintain the TRAIL-resistant phenotype, thus making these miRs as promising therapeutic targets for TRAIL resistance in glioma.
Subject(s)
Apoptosis/drug effects , MicroRNAs/genetics , TNF-Related Apoptosis-Inducing Ligand/pharmacology , 3' Untranslated Regions/genetics , Blotting, Northern , Blotting, Western , Caspase 3/genetics , Caspase 3/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioma/genetics , Glioma/metabolism , Glioma/pathology , HEK293 Cells , Humans , MicroRNAs/metabolism , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Cells, Cultured , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Glioblastoma is the most frequent brain tumor in adults and is the most lethal form of human cancer. Despite the improvements in treatments, survival of patients remains poor. In order to identify microRNAs (miRs) involved in glioma tumorigenesis, we evaluated, by a miRarray, differential expression of miRs in the tumorigenic glioma LN-18, LN-229 and U87MG cells compared with the non-tumorigenic T98G cells. Among different miRs we focused our attention on miR-221 and -222. We demonstrated the presence of a binding site for these two miRs in the 3' untranslated region of the protein tyrosine phosphatase µ (PTPµ). Previous studies indicated that PTPµ suppresses cell migration and is downregulated in glioblastoma. Significantly, we found that miR-221 and -222 overexpression induced a downregulation of PTPµ as analyzed by both western blot and real-time PCR. Furthermore, miR-222 and -221 induced an increase in cell migration and growth in soft agar in glioma cells. Interestingly, the re-expression of PTPµ gene was able to revert the miR-222 and -221 effects on cell migration. Furthermore, we found an inverse correlation between miR-221 and -222 and PTPµ in human glioma cancer samples. In conclusion, our results suggest that miR-221 and -222 regulate glioma tumorigenesis at least in part through the control of PTPµ protein expression.
Subject(s)
Brain Neoplasms/genetics , Glioblastoma/genetics , MicroRNAs/genetics , Receptor-Like Protein Tyrosine Phosphatases, Class 2/genetics , 3' Untranslated Regions/genetics , Animals , Binding Sites/genetics , Blotting, Western , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Movement , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , In Situ Hybridization , Mice , Mice, Nude , MicroRNAs/metabolism , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Oligonucleotide Array Sequence Analysis , Receptor-Like Protein Tyrosine Phosphatases, Class 2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Heterologous , Tumor Burden/geneticsABSTRACT
Akt is a serine-threonine kinase that has an important role in transducing survival signals. Akt also regulates a number of proteins involved in the apoptotic process. To find new Akt interactors, we performed a two-hybrid screening in yeast using full-length Akt cDNA as bait and a human cDNA heart library as prey. Among 200 clones obtained, two of them were identified as coding for the c-FLIP(L) protein. c-FLIP(L) is an endogenous inhibitor of death receptor-induced apoptosis through the caspase-8 pathway. Using co-immunoprecipitation experiments of either transfected or endogenous proteins, we confirmed the interaction between Akt and c-FLIP(L). Furthermore, we observed that c-FLIP(L) overexpression interferes with Gsk3-ß phosphorylation levels. Moreover, through its effects on Gsk3ß, c-FLIP(L) overexpression in cancer cells induced resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This effect was mediated by the regulation of p27(Kip1) and caspase-3 expression. These results indicate the existence of a new mechanism of resistance to TRAIL in cancer cells, and unexpected functions of c-FLIP(L).
